[Odontoclastic resorption lesions (ORL) and chronic gingivitis stomatitis in cats].
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Animals↗
Biomedical subjects
H Vrieling
Publications and source records attributed to H Vrieling.
Assays to predict the genotoxicity of the chromosomal mutagen etoposide -- focussing on the best assay.
The topoisomerase II inhibitor etoposide is used routinely to treat a variety of cancers in patients of all ages. As a result of its extensive use in the clinic and its association with secondary malignancies it has become a compound of great interest with regard to its genotoxic activity in vivo. This paper describes a series of assays that were employed to determine the in vivo genotoxicity of etoposide in a murine model system. The alkaline comet assay detected DNA damage in the bone marrow mononuclear compartment over the dose range of 10--100mg/kg and was associated with a large and dose dependent rise in the proportion of cells with severely damaged DNA. In contrast, the bone marrow micronucleus assay was found to be sensitive to genotoxic damage between the doses of 0.1--1mg/kg without any corresponding increases in cytotoxicity. An increase in the mutant frequency was undetectable at the Hprt locus at administered doses of 1 and 10mg/kg of etoposide, however, an increase in the mutant frequency was seen at the Aprt locus at these doses. We conclude that the BMMN assay is a good short-term predictor of the clastogenicity of etoposide at doses that do not result in cytotoxic activity, giving an indication of potential mutagenic effects. Moreover, the detection of mutants at the Aprt locus gives an indication of the potential of etoposide to cause chromosomal mutations that may lead to secondary malignancy.
Adenine Phosphoribosyltransferase↗
Gene and sequence specificity of DNA damage induction and repair: consequences for mutagenesis.
The field of DNA repair has been expanded enormously in the last 20 years. In this paper, work on gene and sequence specificity of DNA damage induction and repair is summarized in the light of the large and broad contribution of Phil Hanawalt to this field of research. Furthermore, the consequences of DNA damage and repair for mutation induction is discussed, and the contribution of Paul Lohman to the development of assays employing transgenic mice for the detection of gene mutations is highlighted.
Animals↗
Loss of heterozygosity in somatic cells of the mouse. An important step in cancer initiation?
Loss of heterozygosity (LOH) of tumour suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Recently, we and others have developed mouse models in which the frequency and nature of LOH events at an autosomal locus can be elucidated in genetically stable normal somatic cells. In this paper, an overview is presented of recent studies in LOH-detecting mouse models. Molecular mechanisms that lead to LOH and the effects of genetic and environmental variables are discussed. The general finding that LOH of a marker gene occurs frequently in somatic cells of the mouse without deleterious effects on cell viability, suggests that also tumour suppressor genes are lost in similar frequencies. LOH of tumour suppressor genes may thus be an initiating event in cancer development.
Animals↗
Mouse mismatch repair gene Msh2 is not essential for transcription-coupled repair of UV-induced cyclobutane pyrimidine dimers.
The human mutS homolog gene MSH2 is essential for DNA mismatch repair (MMR) and defects in this gene can result in increased mutagenesis, genomic instability and hereditary nonpolyposis colorectal cancer (HNPCC). Besides correcting mismatch errors arising from DNA replication, it was shown that deficiencies in bacterial and human MMR genes including MSH2 resulted in defective transcription-coupled repair (TCR) of UV-induced photolesions. Here we show that MMR-deficient fibroblasts derived from two independent isogenic mouse strains with defined Msh2 deficiencies are as proficient in TCR of UV-induced cyclobutane pyrimidine dimers (CPD) as wildtype fibroblasts. Our results indicate that in mouse cells Msh2 is not essential for TCR of UV-induced CPD in contrast to bacteria and human cells and suggest that the biological effects of UV in mouse Msh2(-/-) cells and mice are not due to defective TCR.
Animals↗
Mitotic maneuvers in the light.
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Adenine Phosphoribosyltransferase↗
DMBA-induced toxic and mutagenic responses vary dramatically between NER-deficient Xpa, Xpc and Csb mice.
Heterogeneity in cancer susceptibility exists between patients with an inherited defect in nucleotide excision repair (NER). While xeroderma pigmentosum (XP) patients have elevated skin cancer rates, Cockayne syndrome (CS) patients do not appear to have increased cancer susceptibility. To investigate whether differences in mutagenesis are the basis for the variability in cancer proneness, we studied mutagenesis at the X-chromosomal Hprt gene and the autosomal Aprt gene in splenic T-lymphocytes after 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure in total NER-deficient Xpa mice, global genome repair (GGR)-deficient Xpc mice and transcription coupled repair (TCR)-deficient Csb mice. Surprisingly, while all intraperitoneally-treated Xpc(-/-) mice survived a dose of 40 mg/kg DMBA, a substantial fraction of the treated Xpa(-/-) and Csb(-/-) mice died a few days after treatment with a 20-fold lower dose. Functional TCR of DMBA adducts in Xpc(-/-) mice thus appears to alleviate DMBA toxicity. However, the mutagenic response in Xpc(-/-) mice was +/- 2-fold enhanced at both the Hprt and the Aprt gene compared to heterozygous controls, indicating that GGR at least partially removes DMBA adducts from the genome overall. DMBA-induced SCE frequencies in mouse dermal fibroblasts were significantly enhanced in Xpa- and Csb-, but not in Xpc-deficient background compared to the frequency in normal fibroblasts. These results indicate that both damage-induced cytotoxicity as well as intra-chromosomal recombinational events were not correlated to differences in cancer susceptibility in human NER syndrome patients.
9,10-Dimethyl-1,2-benzanthracene↗
The relationship between benzo[a]pyrene-induced mutagenesis and carcinogenesis in repair-deficient Cockayne syndrome group B mice.
Cockayne syndrome (CS) patients are deficient in the transcription coupled repair (TCR) subpathway of nucleotide excision repair (NER) but in contrast to xeroderma pigmentosum patients, who have a defect in the global genome repair subpathway of NER, CS patients do not have an elevated cancer incidence. To determine to what extent a TCR deficiency affects carcinogen-induced mutagenesis and carcinogenesis, CS group B correcting gene (CSB)-deficient mice were treated with the genotoxic carcinogen benzo(a)pyrene (B[a]P) at an oral dose of 13 mg/kg body weight, three times a week. At different time points, mutant frequencies at the inactive lacZ gene (in spleen, liver, and lung) as well as at the active hypoxanthine phosphoribosyltransferase (Hprt) gene (in spleen) were determined to compare mutagenesis at inactive versus active genes. B[a]P treatment gave rise to increased mutant frequencies at lacZ in all of the organs tested without a significant difference between CSB-/- and wild-type mice, whereas B[a]P-induced Hprt mutant frequencies in splenic T-lymphocytes were significantly more enhanced in CSB-/- mice than in control mice. The sequence data obtained from Hprt mutants indicate that B[a]P adducts at guanine residues were preferentially removed from the transcribed strand of the Hprt gene in control mice but not in CSB-/- mice. On oral treatment with B[a]P, the tumor incidence increased in both wild-type and CSB-deficient animals. However, no differences in tumor rate were observed between TCR-deficient CSB-/- mice and wild-type mice, which is in line with the normal cancer susceptibility of CS patients. The mutagenic response at lacZ, in contrast to Hprt, correlated well with the cancer incidence in CSB-/- mice after B[a]P treatment, which suggests that mutations in the bulk of the DNA (inactive genes) are a better predictive marker for carcinogen-induced tumorigenesis than mutations in genes that are actively transcribed. Thus, the global genome repair pathway of NER appears to play an important role in the prevention of cancer.
Animals↗
Age-dependent spontaneous mutagenesis in Xpc mice defective in nucleotide excision repair.
DNA damages caused by cellular metabolites and environmental agents induce mutations, that may predispose to cancer. Nucleotide excision repair (NER) is a major cellular defence mechanism acting on a variety of DNA lesions. Here, we show that spontaneous mutant frequencies at the Hprt gene increased 30-fold in T-lymphocytes of 1 year old Xpc-/- mice, possessing only functional transcription-coupled repair (TCR). Hprt mutant frequencies in Xpa-/- and Csb-/- mice that both have a defect in this NER subpathway, remained low during ageing. In contrast to current models, the elevated mutation rate in Xpc-/- mice does not lead to an increased tumour incidence or premature ageing. Oncogene (2000) 19, 5034 - 5037
Aging↗
UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II.
Cells from patients with Cockayne syndrome (CS) are hypersensitive to DNA-damaging agents and are unable to restore damage-inhibited RNA synthesis. On the basis of repair kinetics of different types of lesions in transcriptionally active genes, we hypothesized previously that impaired transcription in CS cells is a consequence of defective transcription initiation after DNA damage induction. Here, we investigated the effect of UV irradiation on transcription by using an in vitro transcription system that allowed uncoupling of initiation from elongation events. Nuclear extracts prepared from UV-irradiated or mock-treated normal human and CS cells were assayed for transcription activity on an undamaged beta-globin template. Transcription activity in nuclear extracts closely mimicked kinetics of transcription in intact cells: extracts from normal cells prepared 1 h after UV exposure showed a strongly reduced activity, whereas transcription activity was fully restored in extracts prepared 6 h after treatment. Extracts from CS cells exhibited reduced transcription activity at any time after UV exposure. Reduced transcription activity in extracts coincided with a strong reduction of RNA polymerase II (RNAPII) containing hypophosphorylated C-terminal domain, the form of RNAPII known to be recruited to the initiation complex. These results suggest that inhibition of transcription after UV irradiation is at least partially caused by repression of transcription initiation and not solely by blocked elongation at sites of lesions. Generation of hypophosphorylated RNAPII after DNA damage appears to play a crucial role in restoration of transcription. CS proteins may be required for this process in a yet unknown way.
Base Sequence↗
Spontaneous and chemically induced point mutations in HPRT cDNA of the metabolically competent human lymphoblastoid cell line, MCL-5.
Thioguanine-resistant clones of the human lymphoblastoid cell, MCL-5, which carries two recombinant plasmids expressing xenobiotic metabolizing enzymes, were obtained spontaneously and after treatment with 0.1 microgram/ml benzo[a]pyrene (BaP), 1.0 microgram/ml 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), and 10 micrograms/ml cigarette smoke condensate (CSC). Treatment with the chemicals reduced the cloning efficiency (CE) of MCL-5 cells from about 30% in untreated cultures to about 10% after treatment with NNK and to about 1% or to less than 1% after treatment with CSC or BaP, respectively. At the same time, the mutant frequencies were increased about sevenfold above those of untreated cultures. Among a total of 138 independent mutant clones that had resulted from 55 separate cultures, 60 point mutations were identified within the hypoxanthine guanine phosphoribosyltransferase (HPRT) reading frame by sequencing full-size reverse transcription polymerase chain reaction (RT-PCR) products from thioguanine-resistant clones. The identified 53 coding errors were distributed among 33 locations and types. Among the 30 types of single basepair substitutions leading to coding errors, 12 had not been described before. In the present set of point mutations, the distribution of base substitution types as well as of mutated sites appeared to be influenced by the treatment with the chemicals. Thus, the ratio of G to T transversions increased from 3 among 19 spontaneous point mutations in the HPRT coding region to 9 among 21 BaP-induced point mutations. The G119T and G208T transversions were found three times each, exclusively after treatment with BaP, while the accumulation of two to eight incidences of the G97T, CG142/3TA, C508T, T583A and G599A mutations was split among different treatments. All eight identified point mutations identified after NNK treatment were at G or T residues on either strand that were followed by additional G or T residues.
Benzo(a)pyrene↗
The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage.
In this study the role of nucleotide excision repair (NER) in protecting mouse embryonic stem (ES) cells against the genotoxic effects of UV-photolesions was analysed. Repair of cyclobutane pyrimidine dimers (CPD) in transcribed genes could not be detected whereas the removal of (6-4) photoproducts (6-4PP) was incomplete, already reaching its maximum (30%) 4 h after irradiation. Measurements of repair replication revealed a saturation of NER activity at UV doses >5 J/m2 while at a lower dose (2.5 J/m2) the repair kinetics were similar to those in murine embryonic fibroblasts (MEFs). Cytotoxic and mutagenic effects of photolesions were determined in ES cells differing in NER activity. ERCC1-deficient ES cells were hypermutable (10-fold) compared to wild-type cells, indicating that at physiologically relevant doses ES cells efficiently remove photolesions. The effect of the NER deficiency on cytoxicity was only 2-fold. Exposure to high UV doses (10 J/m2) resulted in a rapid and massive induction of apoptosis. Possibly, to avoid the accumulation of mutated cells, ES cells rely on the induction of a strong apoptotic response with a simultaneous shutting down of NER activity.
Animals↗
Modulation of the toxic and mutagenic effects induced by methyl methanesulfonate in Chinese hamster ovary cells by overexpression of the rat N-alkylpurine-DNA glycosylase.
Exposure of mammalian cells to alkylating agents causes transfer of alkyl groups to N- as well as O-atoms of DNA bases. Especially the O-alkylated G and T bases have strong mutagenic properties, since they are capable of mispairing during replication. The mutagenic potential of N-alkylbases is less clear although specific base excision repair (BER) pathways exist which remove those lesions from the DNA. We investigated the relative contribution of N-alkylations to mutation induction at the Hprt gene in cultured Chinese hamster ovary cells (CHO). To this end BER activity in CHO cells was modulated by introduction of an expression vector carrying the rat N-alkylpurine-DNA glycosylase (APDG) gene, which codes for a glycosylase that is able to remove 3-methyladenine and 7-methylguanine from DNA thereby generating apurinic sites. Upon selection of a CHO clone which 10 times overproduced APDG compared to control CHO cells, mutation induction, the mutational spectrum, and cell survival were determined in both cell lines following treatment with methyl methanesulfonate (MMS). The results show that over-expression of APDG renders CHO cells more sensitive for mutation induction as well as cytotoxicity induced by MMS. The involvement of apurinic sites in induction of base pair changes at positions where 3-methyladenine was induced is inferred from the observation that the mutational spectrum of MMS-induced mutations in APDG-CHO cells showed twice as much base pair changes at AT base pairs (33.3%) compared to the spectrum of MMS-induced mutations in CHO-control cells (15.8%).
Adenine↗
Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo.
The butylating agent N-n-butyl-N-nitrosourea (BNU) was employed to study the role of nucleotide excision repair (NER) in protecting mammalian cells against the genotoxic effects of monofunctional alkylating agents. The direct acting agent BNU was found to be mutagenic in normal and XPA mouse splenocytes after a single i.p. treatment in vivo. After 25 and 35 mg/kg BNU, but not after 75 mg/ kg, 2- to 3-fold more hprt mutants were detected in splenocytes from XPA mice than from normal mice. Using O6-alkylguanine-DNA alkyltransferase (AGT)-deficient hamster cells, it was found that NER-deficient CHO UV5 cells carrying a mutation in the ERCC-2 gene were 40% more mutable towards lesions induced by BNU when compared with parental NER-proficient CHO AA8 cells. UV5 cells were 1.4-fold more sensitive to the cytotoxic effects of BNU compared with AA8 cells. To investigate whether this increased sensitivity of NER-deficient cells is modulated by AGT activity, cell survival studies were performed in human and mouse primary fibroblasts as well. BNU was 2.7-fold more toxic for mouse XPA fibroblasts compared with normal mouse fibroblasts. Comparable results were found for human fibroblasts. Taken together these data indicate that the role of NER in protecting rodent cells against the mutagenic and cytotoxic effects of the alkylating agent BNU depends on AGT.
Animals↗
Mutation in mammalian cells in vitro and in vivo as indicators of exposure to carcinogens.
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Animals↗
Heterozygous Aprt mouse model: detection and study of a broad range of autosomal somatic mutations in vivo.
During the development of cancer a series of specific genetic alterations have to occur in a stepwise fashion to transform a normal somatic cell into a malignant tumor cell. These genetic changes can be roughly divided in two groups: mutations in proto-oncogenes that result in a constantly activated gene product and mutations in tumor-suppressor genes that result in loss of function. While oncogenic mutations often have a dominant phenotype and mutation of one allele is sufficient for activation, in general both alleles of a tumor suppressor gene have to be disrupted to abolish its function. The requested specificity for activating mutations in proto-oncogenes is high, since only a limited number of mutations at specific sites result in an activated protein. In contrast, disruption of a tumor suppressor gene can be accomplished via various mechanisms. Familial cancers often contain a germline mutation in one allele of a tumor suppressor gene. In tumors, the second allele is then frequently lost by genetic alterations that also affect the heterozygous state of multiple loci adjacent to the tumor suppressor gene. Genetic events especially, such as mitotic recombination, chromosome loss and deletion, are frequently responsible for the loss of the functional allele of heterozygous mutant tumor suppressor genes. We generated an Aprt(+/-) mouse model that allows us to study in detail the nature of the alterations that lead to loss of the wild-type Aprt allele in somatic cells. These genetic changes are thought to be analogous to those occurring at autosomal tumour suppressor genes, where they may contribute to the development of cancer. Furthermore, this mouse model allows determination of the extent and mechanisms by which chemical carcinogens induce loss of heterozygosity and identification of the nature of the DNA adducts responsible.
9,10-Dimethyl-1,2-benzanthracene↗
Carcinogen-induced loss of heterozygosity at the Aprt locus in somatic cells of the mouse.
Genetic events leading to the loss of heterozygosity (LOH) have been shown to play a crucial role in the development of cancer. However, LOH events do not occur only in genetically unstable cancer cells but also have been detected in normal somatic cells of mouse and man. Mice, in which one of the alleles for adenine phosphoribosyltransferase (Aprt) has been disrupted by gene targeting, were used to investigate the potency of carcinogens to induce LOH in vivo. After 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure, a 3-fold stronger mutagenic response was detected at the autosomal Aprt gene than at the X chromosomal hypoxantine-guanine phosphoribosyltransferase (Hprt) gene in splenic T-lymphocytes. Allele-specific PCR analysis showed that the normal, nontargeted Aprt allele was lost in 70% of the DMBA-induced Aprt mutants. Fluorescence in situ hybridization analysis demonstrated that the targeted allele had become duplicated in almost all DMBA-induced mutants that displayed LOH at Aprt. These results indicate that the main mechanisms by which DMBA caused LOH were mitotic recombination or chromosome loss and duplication but not deletion. However, after treatment with the alkylating agent N-ethyl-N-nitrosourea, Aprt had a similar mutagenic response to Hprt while the majority (90%) of N-ethyl-N-nitrosourea-induced Aprt mutants had retained both alleles. Unexpectedly, irradiation with x-rays, which induce primarily large deletions, resulted in a significant increase of the mutant frequency at Hprt but not at Aprt. This in vivo study clearly indicates that, in normal somatic cells, carcinogen exposure can result in the induction of LOH events that are compatible with cell survival and may represent an initiating event in tumorigenesis.
9,10-Dimethyl-1,2-benzanthracene↗
Determination of spontaneous loss of heterozygosity mutations in Aprt heterozygous mice.
A mouse model was generated to investigate loss of heterozygosity (LOH) events in somatic cells. The adenine phosphoribosyltransferase ( Aprt ) gene was disrupted in embryonic stem cells using a conventional gene targeting approach and subsequently Aprt hetero-zygous and homozygous mice were derived. Aprt homozygous deficient animals were viable though the mendelian inheritance pattern was skewed. On average these mice died at 6 months of age from severe renal failure. In T-lymphocytes of Aprt heterozygous mice the mean spontaneous mutant frequency at the Aprt locus was 8.7 x 10(-6) while the frequency was 0.8 x 10(-6) at the hypoxanthine phosphoribosyltransferase locus. In order to determine whether LOH events contribute to the high spontaneous mutant frequency at the Aprt locus, 140 Aprt mutant T-lymphocyte clones were expanded and analysed by allele-specific PCR. In 97 (69%) of these clones the wild-type allele had been lost. Nine of the mutant clones were characterized in more detail using dual-coloured fluorescence in situ hybridization analysis. Five out of six of the mutant clones which arose from an LOH event, based on the PCR assay, contained a duplication of the targeted allele. Therefore, mitotic recombination or chromosome loss followed by duplication of the remaining homologue appears to be the predominant mechanism for the in vivo generation of Aprt mutant T-lymphocytes.
Adenine Phosphoribosyltransferase↗