Biomedical subjects

C Schmutte

Publications and source records attributed to C Schmutte.

At least 19 recordsLinked to original sources

PubMed2001

The interaction of DNA mismatch repair proteins with human exonuclease I.

Exonucleolytic degradation of DNA is an essential part of many DNA metabolic processes including DNA mismatch repair (MMR) and recombination. Human exonuclease I (hExoI) is a member of a family of conserved 5' --> 3' exonucleases, which are implicated in these processes by genetic studies. Here, we demonstrate that hExoI binds strongly to hMLH1, and we describe interaction regions between hExoI and the MMR proteins hMSH2, hMSH3, and hMLH1. In addition, hExoI forms an immunoprecipitable complex with hMLH1/hPMS2 in vivo. The study of interaction regions suggests a biochemical mechanism of the involvement of hExoI as a downstream effector in MMR and/or DNA recombination.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Characterization of the human Rad51 genomic locus and examination of tumors with 15q14-15 loss of heterozygosity (LOH).

Human Rad51 (hRad51) has been found to be associated with BRCA1, BRCA2, and p53 either directly or indirectly and is one of at least eight human genes that are members of the Escherichia coli RecA/Saccharomyces cerevisiae Rad51 family thought to affect genomic stability through DNA recombination/repair processes. While inactivation of DNA mismatch repair clearly leads to instability of repeated sequences and to an increased risk for tumorigenesis, such a parallel for the RecA family members has not been reported. Recently, a high frequency of loss of heterozygosity at chromosome 15q14-15, near the genomic region containing hRad51, has been reported in human tumors (W. Wick et al., Oncogene, 12: 973-978, 1996). To determine whether hRad51 inactivation may be involved in the etiology of these tumors, we have characterized the hRad51 genetic locus and mapped it to chromosome 15q14-15 within the central region of loss of heterozygosity. However, single-strand conformational polymorphism analysis and direct sequencing of tumors did not reveal any mutations in the hRad51 coding sequence or intron/exon boundaries. We also examined the DNA methylation status of a CpG-rich region in the putative hRad51 promoter region. No indication of hypermethylation was found. These results suggest that hRad51 is not a tumor suppressor because it is either an essential gene, redundant gene and/or independent of the BRCA1/BRCA2 tumor suppressor pathway(s).

5' Untranslated Regions↗

PubMed1998

Refined chromosomal localization of the mismatch repair and hereditary nonpolyposis colorectal cancer genes hMSH2 and hMSH6.

The genomic loci for the mismatch repair genes hMSH2 and hMSH6 were mapped by fluorescence in situ hybridization, analysis of radiation hybrid panel markers, and linkage analysis of syntenic chromosome regions between human and mouse. Both genes were localized to chromosome 2p21, adjacent to the luteinizing hormone/choriogonadotropin receptor gene (LHCGR; 2p21), telomeric to the D2S123 polymorphic marker, and centromeric to the calmodulin-2 gene (CALM-2; 2p22-21) and son-of-sevenless gene (SOS; 2p22-21). The genomic locations of hMSH2 and hMSH6 appears to be within 1 Mb of each other because they could not be separated by interphase fluorescence in situ hybridization. These results clarify the position of the chromosome 2 hereditary nonpolyposis colorectal cancer locus, which was originally reported to be associated with an adjacent region (chromosome 2p14-16).

Animals↗

PubMed1998

Human exonuclease I interacts with the mismatch repair protein hMSH2.

DNA mismatch repair (MMR) plays a vital role in the faithful replication of DNA, and its inactivation leads to a mutator phenotype that has been associated with the common cancer susceptibility syndrome Hereditary Non-Polyposis Colorectal Cancer (HNPCC). Here, we report on a novel human exonuclease (hExoI) that is related to the yeast exonuclease 1. The hExoI cDNA comprises 2541 bp, which code for a Mr 94,000 protein that appears to be highly expressed in testis tissue and at very low levels in other tissues. The hExoI gene has 14 exons and is located on chromosome 1q43, as determined by fluorescence in situ hybridization and radiation hybrid mapping. hExoI was found to interact strongly with the human MMR protein hMSH2, suggesting its involvement in the MMR process and/or DNA recombination.

Amino Acid Sequence↗

PubMed1997

Human thymine-DNA glycosylase maps at chromosome 12q22-q24.1: a region of high loss of heterozygosity in gastric cancer.

Spontaneous hydrolytic deamination of 5-methylcytosine leads to T:G mismatches in double-stranded DNA and comprises a major threat for the integrity of both the DNA primary sequence as well as the epigenetic information stored in the DNA methylation pattern. Failure of the cellular DNA repair machinery to recognize and repair such mismatched nucleotides can lead to a mutator phenotype and subsequent carcinogenesis. A thymine-DNA glycosylase (TDG) has been described that initiates T:G mismatch repair by specifically excising the mismatched T. We have studied the TDG genomic locus and the expression of this enzyme to evaluate its role in cancer development. TDG is highly expressed in thymus and is expressed at lower levels in all human tissues analyzed. The TDG gene has 10 exons covering a region of >25 kb and is located on chromosome 12q22-q24.1. Because gastric tumors have been shown to contain a high percentage of C-->T mutations at CpG sites, we used a microsatellite found in intron 8 of the TDG locus to screen gastric tumor samples for loss of heterozygosity. Although our analysis showed loss of heterozygosity in 10 of 24 samples (42%), none of those tumor samples revealed a mutation in the coding sequence of the remaining TDG allele as analyzed by single-strand conformational polymorphism. Expression of the TDG was not determined because of the limited availability of RNA in these primary tumor samples. At present, we have found no evidence that TDG is central to the development of gastric cancer, limiting the importance of TDG in T:G mismatch repair and subsequent carcinogenesis.

Base Sequence↗

PubMed1997

Alterations in DNA methylation are early, but not initial, events in ovarian tumorigenesis.

We compared global levels of DNA methylation as well as methylation of a specific locus (MyoD1) in ovarian cystadenomas, ovarian tumours of low malignant potential (LMP) and ovarian carcinomas to investigate the association between changes in DNA methylation and ovarian tumour development. As we realized that cystadenomas showed different methylation patterns from both LMP tumours and carcinomas, we verified their monoclonal origin as a means of confirming their true neoplastic nature. High-pressure liquid chromatographic (HPLC) analyses showed that global methylation levels in LMP tumours and carcinomas were 21% and 25% lower than in cystadenomas respectively (P = 0.0001 by one-way variance analysis). Changes in the methylation status of the MyoD1 locus were not seen in any of ten cystadenomas analysed but were present in five of ten LMP tumours and in five of ten carcinomas (P = 0.03). These findings suggest that alterations in DNA methylation are absent (or at least not as extensive) in ovarian cystadenomas, but are present in LMP tumours, the phenotypic features of which are intermediate between those of benign and malignant ovarian tumours. The results also emphasize the merit of distinguishing ovarian LMP tumours from cystadenomas, in spite of their similar clinical characteristics.

Blotting, Southern↗

PubMed1997

Presence of p53 mutations in primary nasopharyngeal carcinoma (NPC) in non-Asians of Los Angeles, California, a low-risk population for NPC.

Mutatins of the p53 tumor suppressor gene are rare in nasopharyngeal carcinoma (NPC) patients who reside in high-risk areas, such as Southeastern China. Among this high-risk group, a pre-existing infection with the EBV and consumption of Cantonese salted fish are closely associated with NPC. We investigated the prevalence of p53 mutations in 28 primary NPC specimens from white (including Hispanic) and African-American patients in Los Angeles, who are at low risk for NPC. Using PCR-based single-strand conformational polymorphism and direct sequencing, we found four mutations (14%) in exons 5-8 of the p53 gene in four patients. All were C-to-T transition mutations: two were present in exon 5-one at codon 142 [CCT (Pro)-->CTT (Leu)] and another at codon 144 [CAG (Gln)-->TAG (stop codon)]. The other two mutations were identified in exon 8: one at codon 273 [CGT (Arg)-->CAT (His)], a CpG site, and one at codon 271, a silent mutation [GAG (Glu)-->GAA (Glu)]. This is the first report investigating the presence of p53 missense mutations in NPC among a low-risk population. Our data indicate that p53 is also an infrequent event among NPC patients at low risk for the disease.

Adolescent↗

PubMed1996

Structure and characterization of the human tissue inhibitor of metalloproteinases-2 gene.

We report here the characterization of the human tissue inhibitor of metalloproteinases-2 (TIMP-2) gene. The gene is 83 kilobase pairs (kb) long with exon-intron splicing sites located in preserved positions among the three members of the TIMP family. A 2.6-kb genomic DNA fragment flanking the 5'-end of the gene contains several regulatory elements including five Sp1, two AP-2, one AP-1, and three PEA-3 binding sites. Despite the presence of a complete AP-1 consensus at position -281, the promoter did not respond to 12-O-tetradecanoylphorbol-13-acetate treatment. However, 12-O-tetradecanoylphorbol-13-acetate response was generated by insertion of a similar AP-1 consensus at position -71, indicating the importance of the positioning of this motif. The promoter contains a typical CpG island; however, methylation of this island did not seem to influence gene expression. Analysis of the 3'-end of the gene revealed that the two mRNAs for TIMP-2 (1.2 and 3.8 kb) differ by the selection of their polyadenylation signal sites, but selection of these sites does not affect RNA stability. In summary, the TIMP-2 gene has several features observed in housekeeping genes, and differs significantly from TIMP-1 and TIMP-3 genes. These differences are likely to explain the specific roles that these inhibitors play in the regulation of matrix metalloproteinases.

Amino Acid Sequence↗

PubMed1996

Mechanisms for the involvement of DNA methylation in colon carcinogenesis.

C --> T transitions at CpG sites are the most prevalent mutations found in the p53 tumor suppressor gene in human colon tumors and in the germline (Li-Fraumeni syndrome). All of the mutational hot spots are methylated to 5-methylcytosine, and it has been hypothesized that the majority of these mutations are caused by spontaneous hydrolytic deamination of this base to thymine. We have previously reported that bacterial methyltransferases induce transition mutations at CpG sites by increasing the deamination rate of C --> U when the concentration of the methyl group donor S-adenosylmethionine (AdoMet) drops below its Km, suggesting an alternative mechanism to create these mutations. Unrepaired uracil pairs with adenine during replication, completing the C --> T transition mutation. To determine whether this mechanism could contribute to the development of human colon cancer, we examined the level of DNA (cytosine-5)-methyltransferase (MTase) expression, the concentration of AdoMet, and the activity of uracil-DNA glycosylase in human colon tissues, and searched for the presence of mutations in the MTase gene. Using reverse transcription-PCR methods, we found that average MTase mRNA expression levels were only 3.7-fold elevated in tumor tissues compared with surrounding normal mucosa from the same patient. Also, no mutations were found in conserved regions of the gene in 10 tumors sequenced. High-performance liquid chromatographic analysis of extracts from the same tissues showed that AdoMet concentrations were not reduced below the Km value for the mammalian enzyme, and the concentration ratio of AdoMet:S-adenosylhomocysteine, the breakdown product of AdoMet and the competitive MTase inhibitor, did not differ significantly. Finally, extracts from the tumor tissue efficiently removed uracil from DNA. Therefore, biochemical conditions favoring a mutagenic pathway of C --> U --> T were not found in a target tissue known to undergo a high rate of C --> T transitions at CpG sites.

Base Sequence↗

PubMed1995

A mutant HpaII methyltransferase functions as a mutator enzyme.

DNA (cytosine-5)-methyltransferases can cause deamination of cytosine when the cofactor S-adenosylmethionine (AdoMet) is limiting and thus function as sequence-specific C-->U mutator enzymes. Here we explored whether mutations causing inactivation of the cofactor binding activity of the HpaII methyltransferase, thus mimicking conditions of limiting AdoMet concentration, could convert a DNA methyltransferase to a C-->U mutator enzyme. We created two mutator enzymes from the HpaII methyltransferase (F38S and G40D) which both showed enhanced cytosine deamination activities in vitro and in vivo. Interestingly, the G:U mispairs generated by these enzymes were not repaired completely in bacteria equipped with uracil-DNA glycosylase-initiated repair machinery, giving rise to a potent mutator phenotype. This is the first report showing the creation of mutator enzymes from a DNA methyltransferase and the demonstration of their mutagenicity in living cells.

Base Sequence↗

PubMed1995

Base excision repair of U:G mismatches at a mutational hotspot in the p53 gene is more efficient than base excision repair of T:G mismatches in extracts of human colon tumors.

Approximately 50% of mutations that inactivate the p53 tumor suppressor gene in the germline and in colon tumors are C to T transitions at methylation sites (CpG sites). These mutations are believed to be caused by an endogenous mechanism and spontaneous deamination of 5-methyl-cytosine to T is likely to contribute significantly to this high mutation rate. The resulting T:G mismatches created by this process have been hypothesized to be less efficiently repaired than U:G mismatches formed by deamination of C. We have, therefore, performed the first study to directly compare rates of T:G versus U:G base excision repair at identical sites observed to be mutated in the p53 gene using extracts of human normal colon mucosa and colon carcinoma tissue. Mismatched U was excised up to 6000-fold more efficiently than T, suggesting that differences in repair efficiencies are the major source of C to T transition mutations at CpG sites in human tissues. The data also suggests that T:G mismatches are repaired by additional mechanisms in human cells.

Base Sequence↗

PubMed1994

Mutagenicity of nitric oxide is not caused by deamination of cytosine or 5-methylcytosine in double-stranded DNA.

Several human tumors of diverse histological origin have a high incidence of C:G to T:A transition mutations at methylated CpG sites in tumor suppressor genes. We used a sensitive genetic assay to examine the ability of nitric oxide (NO), a physiological intra- and intercellular messenger molecule, to promote these transitions by deaminating cytosine (C) or methylcytosine (5mC) in double-stranded DNA. Exposure of a test double-stranded plasmid containing C or 5mC at the target site to NO in phosphate-buffered solution at pH 7.4 followed by transformation into Escherichia coli ung- strain to avoid repair of U did not result in a significant increase in reversion frequency. In addition, exposure of E. coli transformed with the target plasmid to an NO-releasing spermine-NO complex during log-phase growth did not result in larger numbers of revertants, whereas Salmonella typhimurium strain TA1535 showed a dose-responsive increase in reversion frequency when treated in the same way. We conclude that genotoxicity of NO is not caused by deamination of C or 5mC to U or T, respectively, in double-stranded DNA. This is supported by the finding that extracts of TA1535 contained high uracil-DNA glycosylase activity, suggesting that the difference in mutagenesis between the strains is not due to a lack of uracil repair. Therefore, mutational hot-spots seen in human tumor tissues at CpG sites are probably not due to the action of NO at 5mC.

5-Methylcytosine↗

PubMed1993

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on protein kinase C and inositol phosphate metabolism in primary cultures of rat hepatocytes.

Adult rat hepatocytes, after maintenance for 24 h in serum-free culture, were treated with the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Short-term treatment (15 min) with TPA, 1 microM, increased protein kinase C (PKC) activity in the particulate fraction of hepatocytes and, concomitantly, decreased the vasopressin (100 nM)-stimulated synthesis of inositol phosphates. The latter effect of TPA could be prevented by prior addition of the PKC inhibitor, H7 (100 microM). After short-term treatment (15 min) with TCDD, 1 pM, no effects on PKC or inositol phosphate metabolism were observed. However, after prolonged exposure to TCDD (3-48 h), the particulate PKC was significantly activated (1.5-fold). In contrast to the effect of TPA (24 h), no down-regulation was found. Moreover, long-term treatment with TCDD significantly enhanced vasopressin-stimulated inositol 1,3,4,5-tetrakisphosphate synthesis, while TPA treatment (24 h) stimulated the synthesis of inositol trisphosphates and inositol 1,3,4,5-tetrakisphosphate. The results suggest that the tumor promoters, TPA and TCDD, act differently on the signal transduction pathways in hepatocytes. Thus, the effects of TCDD on PKC and inositol phosphate metabolism might be mediated by a yet unknown mechanism rather than by direct activation of PKC as seen with TPA.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Formation of genotoxic metabolites from anthraquinone glycosides, present in Rubia tinctorum L.

Rubia tinctorum L., a medicinal plant used for the treatment of kidney and bladder stones, contains a characteristic spectrum of 9,10-anthraquinone derivatives, which are substituted in only one of the aromatic benzo rings. The majority of the anthraquinones present in the plant itself or in plant extracts are glycosides. We investigated the metabolism of two such glycosides, alizarinprimeveroside (AlP) and lucidinprimeveroside (LuP). AlP given orally to rats was metabolized to alizarin (Al) and 1-hydroxyanthraquinone (1-HA). The reductive cleavage of AlP was also observed after treatment of this compound with rat liver enzymes (S9) and NADPH. 1-HA has been reported to induce unscheduled DNA synthesis (UDS) in primary rat hepatocytes (PRH) and intestinal and liver tumors in rats after chronic treatment. The in vitro genotoxicity of 1-HA was confirmed by our present investigations. We also observed that the glycoside AlP was active at inducing UDS in PRH, but the compound was inactive in the Salmonella/microsome assay. Oral administration of LuP to rats resulted in the excretion of lucidin and rubiadin. When LuP was treated with rat liver extract and NADPH, the compound was reduced to rubiadinprimeveroside (RuP), which was hydrolyzed to rubiadin. We have recently shown that lucidin is highly genotoxic in a battery of short-term tests. We now report that rubiadin is also highly genotoxic in Salmonella typhimurium. However, in contrast to lucidin, it requires metabolic activation. In the UDS assay in PRH, rubiadin was even more potent than lucidin and equal to the positive control DMBA. In addition, the glycoside LuP is active in the Salmonella/microsome assay as well as in the UDS assay. The present work demonstrates that the uptake of the anthraquinone glycosides AlP and LuP leads to the rodent carcinogen 1-HA, and to the highly genotoxic compounds lucidin and rubiadin. This extends our previous studies and supports our suggestion that the therapeutic use of Rubia tinctorum may involve a carcinogenic risk.

Animals↗

PubMed1990

Hydroxyanthraquinones as tumor promoters: enhancement of malignant transformation of C3H mouse fibroblasts and growth stimulation of primary rat hepatocytes.

Because danthron, though carcinogenic, does not seem to be genotoxic, it and 8 other hydroxyanthraquinones were comparatively investigated for activities associated with tumor promotion, such as stimulation of cell proliferation and enhancement of malignant transformation. The in vivo treatment of primary rat hepatocytes with danthron, aloe-emodin, chrysophanol, and rhein resulted in a 2-3-fold increase of DNA synthesis, lucidin and purpurin were less active, and emodin, purpuroxanthin, and alizarin were essentially inactive. In addition, danthron, rhein, and chrysophanol (preliminary data), but not alizarin, enhanced transformation of C3H/M2 mouse fibroblasts initiated by N-methyl-N'-nitro-N-nitrosoguanidine or 3-methylcholanthrene. The results of these in vitro studies suggest that hydroxyanthraquinones, possessing 2 hydroxy groups in the 1,8-positions, e.g., danthron, rhein, and chrysophanol, may have tumor-promoting activities. This conclusion is in accordance with the hypothesis that the in vivo carcinogenic activity of danthron may be associated with tumor promotion.

Animals↗

PubMed1990

[The effect of Ca++ antagonists on cellular lipid metabolism].

The antiatherogenic effects of Ca2(+)-antagonists have been proved in animal studies and in man. It is suggested that drugs of this class--unlike lipid-lowering drugs--do not exert their effects by decreasing lipoprotein plasma levels but by a modulation of signal transducers. Ca2(+)-antagonists inhibit the extracellular matrix synthesis and thereby decrease cell adhesion and the modification of matrix-bound low density lipoproteins (LDL). Thus less modified LDL are produced, which are catabolized by scavenging or by phagocytosis of macrophages, leading to a reduction of foam cell production. Ca2(+)-antagonists also enhance the synthesis of membrane phospholipids, e.g. sphingomyelin (SPM), thereby increasing the membrane turnover and fluidity. In addition they positively influence the formation and catabolism of radicals. The resulting membrane protective effects possibly delay the general ageing process and improve the integrity of the cytoskeleton as well as of the adjacent membranes. In various studies it has been established that the use of Ca2(+)-antagonists leads to a reversal of atherosclerotic processes at the stage of early lesions. The effects of this class of drugs on a molecular level will be a focal point of future research. Of special importance will be the study of the cell specific signal transducing processes in those types of cells involved in atherosclerosis and the understanding of their role in the process as a whole.

Arteriosclerosis↗

PubMedDate not supplied

Growth stimulation of primary rat hepatocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The modulation of liver growth control by the tumor promoter, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was investigated in primary hepatocytes of adult rats. Under defined conditions in serum-free cultures, the interaction of TCDD with growth-related hormones was studied. TCDD-treatment of the cultured hepatocytes for two days caused a transient stimulation of both DNA synthesis and mitotic activity. This effect was maximal at the very low nontoxic concentration of 10(-12) M TCDD, i.e., two orders of magnitude below the optimal concentrations for induction of drug metabolizing enzymes. Growth stimulation by TCDD was dependent on the presence of growth-related hormones; in primary rat hepatocytes, TCDD acted synergistically with insulin and epidermal growth factor (EGF) and antagonized the growth inhibition by dexamethasone. Under culture conditions allowing high rates of DNA synthesis, e.g., at low concentrations of dexamethasone, in the presence of EGF plus alpha 1-adrenergic agonists or rat serum, no significant effect of TCDD on cellular growth was observed. Furthermore, TCDD failed to stimulate DNA synthesis in a rat hepatoma cell line, H4IIE, which is less sensitive to growth controlling factors than normal hepatocytes. Therefore, the results suggest that the growth modulation of primary rat hepatocytes by TCDD is the most sensitive parameter of the agent thus far observed. This effect may involve both a release from the growth inhibition caused, for instance, by glucocorticoids, as well as a direct growth-stimulating effect, synergistic to the one induced by insulin.

Adrenergic alpha-Agonists↗

PubMedDate not supplied

Involvement of DNA methylation in human carcinogenesis.

It is now generally accepted that the presence of 5-methylcytosine (5mC) in human DNA has both a genetic and an epigenetic effect on cellular development, differentiation and transformation. First, 5mC is more unstable than its unmethylated counterpart cytosine. Hydrolytic deamination of 5mC leads to a G/T mismatch and subsequently, if unrepaired, to a C-->T transition mutation. Sites of DNA methylation are mutational hotspots in many human tumors. Second, DNA methylation of promoter regions is often correlated with the down regulation of the corresponding gene. Both of these effects have fundamental consequences for basic functions of the cell like cellular differentiation, the development of cancer and possibly other diseases, and on the evolutionary process. Recent hypotheses also propose a role for methylation in the process of aging. In this review we will describe recent findings and hypotheses about the function of 5mC in DNA with the focus on its involvement in human carcinogenesis.

Animals↗