[Ethical and social issues of human genome analysis].
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DNA↗
Biomedical subjects
H Takebe
Publications and source records attributed to H Takebe.
Postlabelling analysis of DNA adducts formed in human hepatoma cells treated with 3-nitrobenzanthrone.
3-Nitrobenzanthrone (NBA) is one of the most mutagenic nitroaromatic compounds that has been found recently in diesel exhaust and airborne particles. A [32P]-postlabelling analysis was carried out to examine the adducts in DNA from human hepatoma HepG2 cells treated with NBA. Two major and two minor adduct spots were obtained in the analysis. The structure of the compound obtained from one of the minor adduct spots was identified to be N-acetyl-3-amino-2-(2'-deoxyguanosin-3', 5'-bisphosphate-8-yl)-benzanthrone, based on identical mobility of the compound with that of synthetic standards in thin-layer chromatography and high performance liquid chromatography. This substance is the identical adduct found in our previous in vitro study. The yet-unidentified major adduct spots may be guanosin- and adenosin-benzanthrone adducts without the N-acetyl group.
Benz(a)Anthracenes↗
Inhibition of X-ray and doxorubicin-induced apoptosis by butyrolactone I, a CDK-specific inhibitor, in human tumor cells.
Cell-cycle progression is coordinately regulated by cyclin-dependent kinases (CDKs). The inhibition of CDKs by p21Waf1/Cip1/Sdi1 prevents the apoptosis of cells treated with DNA-damaging agents. In this study, we found that butyrolactone I, a specific inhibitor of CDC2 family kinases, blocks the X-ray- or doxorubicin-induced apoptosis of DLD1 (p21+/+) human colorectal carcinoma cells in a dose-dependent manner. We also found that butyrolactone I inhibits the CDK2 activity and enhances cell survival after an X-ray irradiation or doxorubicin treatment in both DLD1 (p21-/-) and DLD1 (p21+/+) cells. These findings suggest that butyrolactone I prevents apoptosis by the direct inhibition of CDK and also, possibly, by CDK-inhibition through p53-independent p21-induction. Our findings indicate that CDK activity is required for DNA-damaging agent-induced apoptosis.
4-Butyrolactone↗
[Bioethics in genetic diagnosis and therapy].
Human genetics, or medical genetics have been rarely taught in most of the medical schools in Japan, as there are only several medical schools with genetics departments among 80 medical schools in Japan. Bioethics has just been becoming an important issue in the medical community in Japan. People hate to be told of hereditary diseases, possibly due to the traditional concept of hereditary diseases as punishment for the evil acts of the ancestors. Recent rapid progress in genetic diagnosis and therapy, however, requires the medical community in Japan to consider the bioethical aspects related to human genetics. We need proper guidelines, and the efforts have been made by the government as well as by the Society for Familial Tumor to propose practical guidelines for human genetics. They may considerably be different from those in the Western countries.
Bioethics↗
Involvement of cyclin-dependent kinases in doxorubicin-induced apoptosis in human tumor cells.
Apoptotic cell death caused by doxorubicin, a chemotherapeutic agent, is suppressed by expression of p21 (waf1/cip1/sdi1), a cyclin-dependent kinase (cdk) inhibitor. To examine cdk activity required for doxorubicin-induced apoptosis, we transfected p21-deficient human tumor DLD1(p21-/-) cells with plasmids carrying wild-type p21 and mutated p21 unable to bind to cdks or proliferating cell nuclear antigen. The apoptosis induced at the G(2)/M phase after doxorubicin treatment was suppressed by transient expression of the p21 with cdk-binding activity but not by the p21 lacking the activity. We also transfected cells with plasmids carrying wild-type, dominant negative and constitutively active mutants of cdk2 or cdk4. The apoptosis was suppressed by transient expression of dominant negative mutants of cdk2 or cdk4. These findings indicate that cdk is involved in the doxorubicin-induced apoptosis pathway.
Antibiotics, Antineoplastic↗
Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells.
Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.
Base Sequence↗
Molecular analysis of mutations induced by a benzene metabolite, p-benzoquinone, in mouse cells using a novel shuttle vector plasmid.
Human population has been continually exposed to benzene which is present in our environment as an essential component of petroleum. p-Benzoquinone (p-BQ) is one of the benzene metabolites and is thought to be an ultimate toxic or carcinogenic substance. For molecular analysis of carcinogen-induced mutations in mouse cells, we constructed a new shuttle vector plasmid pNY200 that has supF gene as a target of the mutations and replicates in mouse and in Escherichia coli cells. In p-BQ-treated pNY200 propagated in mouse cells, base substitutions were induced predominantly at G:C sites, and the major mutation was G:C-->A:T transition. Many tandem base substitutions were also induced at CC:GG sequences. By a postlabeling analysis and a polymerase stop assay, we confirmed that p-BQ adducts formed in DNA and mutation sites roughly correspond to the sites where the adducts were formed. Comparing data of pNY200 in mouse cells with those of the similar shuttle vector plasmid pMY189 in human cells should be important for extrapolation of data from mouse to human, because carcinogenicity of chemicals is tested in mice.
Animals↗
Phylogeny of Japanese papilionid butterflies inferred from nucleotide sequences of the mitochondrial ND5 gene.
Phylogenetic relationships among the Japanese papilionid butterflies were analyzed by comparing 783 nucleotide sequences of the mitochondrial gene encoding NADH dehydrogenase subunit 5 (ND5). Phylogenetic trees of the representative species from each family in the superfamily Papilionoidea revealed that the species of the family Papilionidae and those of all other families formed distinct clusters, with a few species of the family Hesperiidae (Hesperioidea) as an outgroup. In the phylogenetic trees of most Japanese species of the family Papilionidae with Nymphalis xanthomelas (Nymphalidae) as an outgroup, the tribe Parnassiini (Parnassiinae) formed a cluster, and the rest formed the other cluster in which the tribe Zerynthiini (Parnassiinae) and the subfamily Papilioninae formed different subclusters. In the Papilioninae cluster, the tribes Troidini and Graphiini formed a subcluster, and the tribe Papilionini formed the other subcluster. These results generally agree with the traditional classification of the papilionid butterflies based on their morphological characteristics and support the proposed evolutionary genealogy of the butterflies based on their morphology, behavior, and larval host plants, except that the tribes Parnasiini and Zerynthiini (both Parnassiinae) are not in the same cluster.
Animals↗
Long-term exposure to a magnetic field (5 mT at 60 Hz) increases X-ray-induced mutations.
Exposure to extremely low frequency magnetic field (ELFMF) at 400 mT has been shown to induce mutations (Mutat. Res., 349: 109-114, 1996; Int. J. Radiat. Biol., 71: 75-79, 1997; and Biochem. Biophys. Res. Commun., 243: 579-584, 1998). However, whether ELFMF at low flux densities (under 1 mT) induces mutations is debatable. We investigated the effect of long-term exposure to 5 mT ELFMF at 60 Hz on mutant frequency. Chinese hamster ovary K1 (CHO-K1) cells were exposed or sham-exposed to 5 mT ELFMF for up to 6 weeks with or without X-irradiation (3 Gy), and the mutant frequency of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene was analyzed. Long-term exposure to 5 mT ELFMF did not increase mutations, suggesting a threshold for mutation induction greater than 115 mA/m2 or a magnetic density of 5 mT. However, enhancement of the X-ray-induced mutation rate was observed after treatment with X-irradiation followed by long-term exposure to 5 mT ELFMF. At little as a 1-week exposure to ELFMF after X-irradiation enhanced the mutation rate. We also found that 400 mT exposure enhanced the mutation rate induced by X-irradiation (Mutat. Res., 349: 109-114, 1996). These results suggest that exposure to more than 5 mT ELFMF may promote X-ray-induced mutations.
Animals↗
Trans-dominant inhibition of poly(ADP-ribosyl)ation potentiates alkylation-induced shuttle-vector mutagenesis in Chinese hamster cells.
In most eukaryotic cells, the catalytic activation of poly(ADP-ribose) polymerase (PARP) represents one of the earliest cellular responses to the infliction of DNA damage. To study the biological function(s) of poly(ADP-ribosyl)ation, we have established stable transfectants (COM3 cells) of the SV40-transformed Chinese hamster cell line C060 which conditionally overexpress the PARP DNA-binding domain upon addition of dexamethasone. We could demonstrate that DNA-binding domain overexpression, which leads to trans-dominant inhibition of poly(ADP-ribosyl)ation, potentiates the cytotoxicity of alkylation treatment and of gamma-radiation. Likewise, carcinogen-induced gene amplification, viewed as a manifestation of genomic instability, was potentiated by the overexpression of the PARP DNA-binding domain. Recently, we studied the effect of trans-dominant PARP inhibition on mutagenesis by employing a shuttle-vector assay in which mutagen-exposed plasmid pYZ289 is electroporated into COM3 cells. We could show that dexamethasone-induced overexpression of the PARP DNA-binding domain in COM3 cells potentiates the mutagenicity of the alkylating agent N-methyl-N-nitrosourea, while no effect of dexamethasone treatment on mutation frequency was recorded in control cells lacking the PARP DNA-binding domain transgene. Taken together, our results further substantiate the role of poly(ADP-ribosyl)ation in the maintenance of genomic integrity and stability under conditions of genotoxic stress.
Alkylating Agents↗
Decreased host-cell reactivation of UV-irradiated adenovirus in human colon tumor cell lines that have normal post-UV survival.
An ongoing study in our laboratories is to examine the relationship of DNA repair defects to human cancer. Our underlying hypothesis has been that human tumors may arise that lack interesting DNA repair pathways if these pathways are important in preventing cancer. In this study, we found that the UV-irradiated adenoviruses showed hypersensitivity when assayed on monolayers of certain human colon tumor cell lines, including three that are reported to have defects in long patch DNA mismatch repair genes and one with no reported defect in mismatch repair. The survival curves showed two components. The first sensitive component was characteristic of 77-95% of the infections depending upon the cell line and the experiment and had an average slope indicating 4.8-fold hypersensitivity to UV. The average of the second-component slopes indicated that the remainder of the infections was accompanied by near-normal repair. Although the value of the first component indicated that the colon tumor lines supported the growth of UV-damaged adenoviruses poorly, the cell lines themselves showed the same post-UV colony-forming ability as did normal human fibroblasts, and their ability to support the growth of N-methyl-N'-nitro-N-nitrosoguanidine-damaged adenoviruses was normal, i.e. it parallelled their ability to repair O6-methylguanine in vitro. We previously observed two-component survival curves when assaying UV-irradiated adenovirus on monolayers of all of seven strains of fibroblasts from Cockayne's syndrome patients. By contrast, single-component curves have been obtained using 21 strains of normal human fibroblasts and seven other tumor lines. We interpret the two-component survival curves in terms of the defective transcription-coupled repair of UV-induced DNA damage that is characteristic both of Cockayne's and certain colon tumor cell lines. In addition, four mismatch repair-deficient colon tumor lines were resistant to killing by elevated levels of dG.
Adenoviruses, Human↗
Molecular analysis of mutations induced by acrolein in human fibroblast cells using supF shuttle vector plasmids.
Types of mutations induced by acrolein in the supF gene on the shuttle vector plasmid pMY189 replicated in normal human fibroblast cells were examined. Base sequence analysis of 92 plasmids with mutations in the supF gene revealed that the majority of the mutations were base substitutions (76%) and the others were deletions and insertions (24%). Single base substitutions were most frequently found (46%), while multiple base substitutions were 18% and tandem (two adjacent) base substitutions were 12% of the mutations. Of the base substitution mutations, G:C to T:A transversions were 44% and G:C to A:T transitions were 24%. The mutations were distributed not randomly but located at several hotspots. Acrolein produced DNA intra-strand cross-links between guanine residues, which might be responsible for rather high induction of the tandem base substitution mutations.
Acrolein↗
Specific tandem GG to TT base substitutions induced by acetaldehyde are due to intra-strand crosslinks between adjacent guanine bases.
Acetaldehyde is present in tobacco smoke and automotive exhaust gases, is produced by the oxidation of ethanol, and causes respiratory organ cancers in animals. We show both the types and spectra of acetaldehyde-induced mutations in supF genes in double- and single-stranded shuttle vector plasmids replicated in human cells. Of the 101 mutants obtained from the double-stranded plasmids, 63% had tandem base substitutions, of which the predominant type is GG to TT transversions. Of the 44 mutants obtained from the single-stranded plasmids, 39% had tandem mutations that are of a different type than the double-stranded ones. The GG to TT tandem substitutions could arise from intra-strand crosslinks. Our data indicate that acetaldehyde forms intra- as well as inter-strand crosslinks between adjacent two-guanine bases. Based upon the following observations: XP-A protein binds to acetaldehyde-treated DNA, DNA excision repair-deficient xeroderma pigmentosum (XP) cells were more sensitive to acetaldehyde than the repair-proficient normal cells, and a higher frequency of acetaldehyde-induced mutations of the shuttle vectors was found in XP cells than in normal cells, we propose that the DNA damage caused by acetaldehyde is removed by the nucleotide excision repair pathway. Since treatment with acetaldehyde yields very specific GG to TT tandem base substitutions in DNA, such changes can be used as a probe to identify acetaldehyde as the causal agent in human tumors.
Acetaldehyde↗
Suppression of high-density magnetic field (400 mT at 50 Hz)-induced mutations by wild-type p53 expression in human osteosarcoma cells.
Exposure of cultured human osteosarcoma cells (Saos-LP-12) to high-density (400 mT at 50 Hz) extremely low frequency magnetic fields (ELFMF) induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene. Saos-LP-12 cells, which are isolated from parental Saos-2 cells and have a deletion in the coding region of the p53 gene, are introduced to the wild-type (wt) p53 expression plasmid (pOPRSVp53). The mutation in Saos-LP-12 cells was suppressed by expression of the introduced wt p53 gene during 400 mT ELFMF exposure. No marked difference in the mutation spectrum was observed among the treatments of ELFMF [p53 (-)], ELFMF [p53 (+)], and sham exposures. Our findings suggest that wt p53 has a function in suppression of DNA replication errors and/or in maintenance of genomic stability after high-density ELFMF exposure.
DNA Mutational Analysis↗
Mutated p21(WAF1/CIP1/SDI1) lacking CDK-inhibitory activity fails to prevent apoptosis in human colorectal carcinoma cells.
Human colorectal tumor cell lines were established which express wildtype p21 or p21 with a mutation at codon 46 (Cys) or 140 (Gly) on IPTG treatment (LacSwitch). The IPTG-induced wildtype p21 bound to CDK2 and PCNA and inhibited CDK activity in the cells and reduced cell growth rate; whereas, both IPTG-induced mutated p21 proteins neither bound to CDK2 nor affected the CDK activity but did bind to PCNA, and they did not affect the cell growth rate. Wildtype p21 suppressed apoptosis and enhanced survival of X-ray-irradiated or adriamycin-treated cells; but, mutated p21 neither suppressed apoptosis nor affected cell survival. When cells were treated with mimosine, a p53-independent p21-inducer, or butyrolactone I, a specific inhibitor of CDK, cellular endogenous p21 was induced and X-ray or adriamycin-induced apoptosis was blocked. These results suggest that CDK-binding or CDK-inhibitory activity of p21 is required to prevent apoptosis, i.e., CDK is required for apoptosis in human tumor cells.
4-Butyrolactone↗
Reduced UV-induced mutations in human osteosarcoma cells stably expressing transfected wild-type p53 cDNA.
We constructed the plasmid which can express human wild-type p53 cDNA and introduced it into the human osteosarcoma cell line SAOS-2 that lacks the chromosomal p53 gene. A cell clone stably expressing p53 protein was isolated and UV sensitivity and UV-induced mutation frequencies of the clone were examined. The UV sensitivity of the clone was slightly higher and UV-induced hprt mutation frequencies of the clone were markedly lower than those of parental SAOS-2 cells. The capability to repair UV-induced DNA damage assessed by the amount of unscheduled DNA synthesis or DNA single strand breaks as well as cell cycle progression after UV irradiation were not different between the clone and SAOS-2 cells. These results indicate that wild-type p53 protein would be involved in the human DNA damage-processing pathway other than the genome-overall excision repair.
Cell Survival↗
Mutations of p16 and p15 tumor suppressor genes and replication errors contribute independently to the pathogenesis of sporadic malignant melanoma.
Mutations of p16 and p15 suppressor oncogenes and the replication errors in six microsatellite loci in sporadic malignant melanomas were analyzed. Four (9.1%) homozygous deletions of both p16 and p15 genes and one point mutation (2.3%) in the p15 gene were detected among 44 primary melanoma samples. One mutation in each of the p16 and p15 genes was observed in ten metastatic lesions. Eight (18.2%) replication errors were detected in three microsatellite loci in the primary melanoma samples, but no replication error was detected in the metastatic samples. None of the samples showed the alteration of p16/p15 genes and the replication errors concomitantly. These results suggest that (1) the homozygous deletions of p16/p15 genes and the replication errors may occur in rather early stages of melanoma tumorigenesis, while the p16/p15 gene mutation may occur in later stages, and (2) the p16 and p15 gene mutations in sporadic malignant melanomas might not be induced by the defect in mismatch repair, implying that p16 as well as p15 gene alterations may play an important role in the pathogenesis of sporadic malignant melanomas.
Adenine Nucleotides↗
Mutagenic specificity of a derivative of 3-nitrobenzanthrone in the supF shuttle vector plasmids.
3-Nitrobenzanthrone (NBA) is a powerful bacterial mutagen and a suspected human carcinogen present in diesel exhaust and airborne particulates [Enya, T., et al. (1997) Environ. Sci. Technol. 31, 2772-2776]. In the accompanying paper [Enya, T., et al. (1998) Chem. Res. Toxcol. 11, 1460-1467], N-acetoxy-N-acetyl-3-aminobenzanthrone (N-Aco-N-Ac-ABA) was synthesized to yield the DNA adducts of NBA. In this work, to investigate the mutagenic specificity of NBA in human cells, we analyzed mutations induced by N-Aco-N-Ac-ABA using the supF shuttle vector plasmids. Base sequence analysis of 110 and 100 plasmids with mutations in the supF gene propagated in normal cells [WI38-VA13] and nucleotide excision repair deficient cells [XP2OS(SV)], respectively, revealed that the majority of the mutations were base substitutions (85 and 90%) and the rest were deletions and insertions (10 and 15%) in both cell lines. About half of the mutant plasmids had a single base substitution. Of the base substitutions, the most frequent mutation was G.C to T.A transversion (41 and 51%), followed by G.C to A.T transitions (18 and 24%) in either cell. The mutations were distributed not randomly but located at several hot spots, and almost all (nine of ten) hot spots were at the sites of G.C base pairs. The polymerase stop assay in the supF gene revealed that N-Aco-N-Ac-ABA preferentially bound to guanine residues, and mutation sites were generally consistent with the sites where the guanine adducts were formed.
Base Sequence↗