Gli1+-PDL Cells Contribute to Alveolar Bone Homeostasis and Regeneration.

The periodontal ligament (PDL) contains mesenchymal stem cells (MSCs) that can differentiate into osteoblasts, cementoblasts, and fibroblasts. Nevertheless, the distribution and characteristics of these cells remain uncertain. Gli1, an essential hedgehog signaling transcription factor, functions in undifferentiated cells during embryogenesis. Therefore, in the present study, the differentiation ability of Gli1+ cells was examined using Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice. In 4-wk-old iGli1/Tomato mice, Gli1/Tomato+ cells were only slightly detected in the PDL, around endomucin-expressing blood vessels. These cells had proliferated over time, localizing in the PDL as well as on the bone and cementum surfaces at day 28. However, in 8-wk-old iGli1/Tomato mice, Gli1/Tomato+ cells were quiescent, as most cells were not immunoreactive for Ki-67. These cells in 8-wk-old mice exhibited high colony-forming unit fibroblast activity and were capable of osteogenic, chondrogenic, and adipogenic differentiation in vitro. In addition, after transplantation of teeth of iGli1/Tomato mice into the hypodermis of wild-type mice, Tomato fluorescence indicating the progeny of Gli1+ cells was detected in the osteoblasts and osteocytes of the regenerated bone. These results demonstrate that Gli1+ cells in the PDL were MSCs and could contribute to the alveolar bone regeneration.

Intermediate length scale organisation in tin borophosphate glasses: new insights from high field correlation NMR.

The structure of tin borophosphate glasses, considered for the development of low temperature sealing glasses or anode materials for Li-batteries, has been analysed at the intermediate length scale by a combination of high field standard and advanced 1D/2D nuclear magnetic resonance techniques. The nature and extent of B/P mixing were analysed using the (11)B((31)P) dipolar heteronuclear multiple quantum coherence NMR sequence and the data interpretation allowed (i) detecting the presence and analysing the nature of the B-O-P linkages, (ii) re-interpreting the 1D (31)P spectra and (iii) extracting the proportion of P connected to borate species. Interaction between the different borate species was analysed using the (11)B double quantum-simple quantum experiment to (i) investigate the presence and nature of the B-O-B linkage, (ii) assign the different borate species observed all along the composition line and (iii) monitor the borate network formation. In addition, (119)Sn static NMR was used to investigate the evolution of the chemical environment of the tin polyhedra. Altogether, the set of data allowed determining the structural units constituting the glass network and quantifying the extent of B/P mixing. The structural data were then used to explain the non-linear and unusual evolution of the glass transition temperature.

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.

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.

[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.

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.

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.

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.

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.

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.

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.

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.

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.

Sensitivity of group F xeroderma pigmentosum cells to UV and mitomycin C relative to levels of XPF and ERCC1 overexpression.

The XPF and ERCC1 proteins form a tight complex and function as an endonuclease to incise on the 5'-side of pyrimidine dimers in DNA. Levels of both proteins are extremely low in group F xeroderma pigmentosum (XP-F) cells. We transfected XP-F cells with the plasmids expressing XPF or ERCC1 and examined levels of both proteins in the cells. Although XP-F cells are sensitive to UV and mitomycin C (MMC), cells overexpressing XPF expressed ERCC1 as well and resistance to UV and MMC was restored to the normal level. In contrast, cells overexpressing ERCC1 did not express XPF and were still sensitive to UV and MMC. These results indicate that both the XPF and ERCC1 proteins are required to repair UV- and MMC-induced DNA damage. Even though a high level of ERCC1, which has been presumed to be a catalytic subunit of the endonuclease, is stably present in XP-F cells, ERCC1 protein alone cannot carry out excision repair completely.

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.

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.

Characterization of molecular defects in xeroderma pigmentosum group F in relation to its clinically mild symptoms.

Xeroderma pigmentosum (XP) complementation group F was first reported in Japan and most XP-F patients reported to date are Japanese. The clinical features of XP-F patients are rather mild, including late onset of skin cancer. Recently a cDNA that corrects the repair deficiency of cultured XP-F cells was isolated. The XPF protein forms a tight complex with ERCC1 and this complex functions as a structure-specific endonuclease responsible for the 5' incision during DNA excision repair. Here we have identified XPF mRNA mutations and examined levels of the mRNA and protein expression in seven primary cell strains from Japanese XP-F patients. The XP-F cell strains were classified into three types in terms of the effect of the mutation on the predicted protein; (i) XPF proteins with amino acid substitutions; (ii) amino acid substituted and truncated XPF proteins; and (iii) truncated XPF protein only. A normal level of expression of XPF mRNA was observed in XP-F cells but XPF protein was extremely low. These results indicate that the detected mutations lead to unstable XPF protein, resulting in a decrease in formation of the ERCC1-XPF endonuclease complex. Slow excision repair of UV-induced DNA damage due to low residual endonuclease activity provides a plausible explanation for the typical mild phenotype of XP-F patients.

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.

Enhanced NOR-1 gene expression by exposure of Chinese hamster cells to high-density 50 Hz magnetic fields.

Enhanced expression of neuron derived orphan receptor (NOR-1) gene was observed by exposure of Chinese hamster ovary K1 (CHO-K1) cells to an extremely low frequency magnetic field (ELFMF) of 50 Hz at 400 mT, but not at 5 mT. The enhanced expression, reaching the maximum at 6 h, was transient and reduced to the control level after exposure to 400 mT ELFMF for 24 h. The NOR-1 expression induced by treatment with forskolin and TPA was further enhanced by the simultaneous treatment with 400 mT ELFMF, in which the maximum response was at 3 h. The NOR-1 expression by these treatments was induced more earlier than that by 400 mT ELFMF alone. When cells were treated with an inhibitor of the protein kinase C (calphostin C or crocetin) and Ca2+ entry blockers (nifedipin and dantrolen) during the 400 mT ELFMF exposure, the enhanced NOR-1 expression was not observed. Exposure of CHO-K1 cells to the high-density 400 mT ELFMF may affect the signal transduction in the cells, resulting in the enhanced NOR-1 gene expression.