Vascular allografts are resistant to methicillin-resistant Staphylococcus aureus through indoleamine 2,3-dioxygenase in a murine model.

OBJECTIVE: Surgical results have shown the superiority of human heart valve and vascular allografts over artificial prostheses when used for the treatment of infectious cardiovascular diseases. However, the mechanism of infection resistance in these allografts has not been determined. In this study the contribution of the inflammatory response after allogeneic transplantation to the antimicrobial mechanism was assessed, focusing on the induction of indoleamine 2,3-dioxygenase, a tryptophan-metabolizing enzyme. METHODS: Aortic transplantation was performed with inbred rats, and aortic allografts, isografts, and control grafts were obtained for the following analyses. The extent of inflammatory-related and indoleamine 2,3-dioxygenase gene expression was measured by means of quantitative reverse transcriptase-polymerase chain reaction, and tryptophan metabolite production in the graft was measured by means of liquid chromatographic/tandem mass spectrometric analysis. The bacteriostatic effect of each graft and tryptophan metabolites was determined by using the methicillin-resistant Staphylococcus aureus proliferation assay. RESULTS: The inflammatory response, including interferon gamma, tumor necrosis factor alpha, and indoleamine 2,3-dioxygenase gene expression, was significant in the allografts but minimal in the isografts and control grafts. Methicillin-resistant S. aureus proliferation was remarkably suppressed when cultured with the allografts but not with the control grafts. Among tryptophan metabolites, the bacteriostatic effect against methicillin-resistant S. aureus was remarkable with 3-hydroxykynurenine, with a minimum inhibitory concentration of 32 mg/L. The 3-hydroxykynurenine level in the allografts was 9-fold greater than that in the control grafts. CONCLUSION: The bacteriostatic effect of the allografts was acquired by inducing indoleamine 2,3-dioxygenase, which resulted in local production of 3-hydroxykynurenine as an antimicrobial agent. This is the first report to document a mechanism of the allograft's infection-resistant property against methicillin-resistant S. aureus growth.

A second-generation profiling system for quantitative methylation analysis of multiple gene promoters: application to lung cancer.

Cancer-specific gene promoter methylation has been described in many types of cancers, and various semi-quantified results have shown their usefulness. Here, we show a more sensitive and specific second-generation system for profiling the DNA methylation status. This method is based on bisulfite reaction of DNA and real-time PCR using two TaqMan MGB probes labeled with different fluorescence, followed by clustering analysis. Primers were designed with CpG-less sequences, and TaqMan MGB probes were designed to contain three or four CpG sites and to be shorter than conventional TaqMan probes. We have added new criteria for primer and probe design for further specificity. We confirmed the reliability of this system and applied it to analysis of lung cancers. Using 10 promoters, 90 primary lung cancers were clustered into six groups consisting of cases having similar smoking status and pathological findings. EGFR mutation and p16 promoter DNA methylation were exclusive, as previously reported; however, DNA methylation in other genes was unrelated to EGFR mutation. This system was also useful to distinguish double primary lung cancers from a single cancer with intrapulmonary metastasis. As above, our system has widespread availability in clinical use and biological research.

Large scale mapping of methylcytosines in CTCF-binding sites in the human H19 promoter and aberrant hypomethylation in human bladder cancer.

The methylation status of binding sites of the insulator protein, CTCF, in the H19 promoter has been suggested as being critical to the regulation of imprinting of the H19/IGF2 locus located in chromosome 11p15. In this study, we have analyzed the methylation of all of seven potential CTCF-binding sites in the human H19 promoter since the methylation status of these sites has not been reported. We found that all the binding sites except the sixth were hypermethylated whereas only the sixth binding site showed allele-specific methylation in normal human embryonic ureteral tissue. We also analyzed the methylation status of these sites in human-mouse somatic-cell-hybrid clones containing a single copy of human chromosome 11 and which were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) to yield clones which expressed human IGF2 and H19 mutually exclusively of each other. In most of the clones, a correlation between methylation of the sixth CTCF-binding site and expression of IGF2 was observed. Therefore, we analyzed the methylation status of this site in human bladder cancer and found hypomethylation of the paternal allele in two of six informative cases. These results demonstrate that only the sixth CTCF-binding site acts as a key regulatory domain for switching between H19 or IGF2 expression, whereas the other sites are not subject to allele-specific methylation. Loss of methylation imprinting of H19 is linked to hypomethylation of the paternal allele in human bladder cancer, unlike the situation in Wilms' tumor and colon cancer where the maternal allele becomes hypermethylated.

Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis.

Aberrantly hypermethylated genes in human lung cancers were searched for by a genome scanning technique, methylation-sensitive-representational difference analysis (MS-RDA). A total of 59 DNA fragments were isolated as those methylated more heavily in either/both of two lung squamous cell carcinoma cell lines, EBC-1 and LK-2, than in a primary culture of normal human bronchial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation was confirmed in primary squamous cell carcinomas, were sequenced. By database searches, 17 of them were shown to be located within 2 kb of putative CpG islands, and five of the 17 DNA fragments had transcribed regions of known genes in their vicinities. By RT-PCR of the five genes in the carcinoma cell lines and NHBE, decreased expression of HTR1B (5-hydroxytryptamine receptor 1B) and EDN1 (endothelin-1) was observed. Sequencing after bisulfite modification showed that the CpG island in the promoter region of HTR1B was hypermethylated, while that of EDN1 was not. Demethylation and re-expression of HTR1B were observed after treatment of LK-2 cells with 5-aza-2'-deoxycytidine. In primary lung cancers, decreased mRNA expression of HTR1B was observed in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohistochemical analysis of endothelin-1 confirmed that its immunoreactivity was reduced in squamous cell carcinoma cells compared with that in normal bronchial epithelial cells. Considering that endothelin-1 induces apoptosis in melanoma cells and that silencing of endothelin receptor B is observed in prostate cancers, its reduced expression was speculated to confer a growth advantage to lung cancer cells. MS-RDA was shown to isolate DNA fragments that are hypermethylated and silenced, such as HTR1B, and those whose expressions are altered and the methylation statuses outside the promoter region are altered, such as EDN1.

The role of DNA methylation in mammalian epigenetics.

Genes constitute only a small proportion of the total mammalian genome, and the precise control of their expression in the presence of an overwhelming background of noncoding DNA presents a substantial problem for their regulation. Noncoding DNA, containing introns, repetitive elements, and potentially active transposable elements, requires effective mechanisms for its long-term silencing. Mammals appear to have taken advantage of the possibilities afforded by cytosine methylation to provide a heritable mechanism for altering DNA-protein interactions to assist in such silencing. Genes can be transcribed from methylation-free promoters even though adjacent transcribed and nontranscribed regions are extensively methylated. Gene promoters can be used and regulated while keeping noncoding DNA, including transposable elements, suppressed. Methylation is also used for long-term epigenetic silencing of X-linked and imprinted genes and can either increase or decrease the level of transcription, depending on whether the methylation inactivates a positive or negative regulatory element.

Hypomethylation of LINE1 retrotransposon in human hepatocellular carcinomas, but not in surrounding liver cirrhosis.

BACKGROUND: Cytosine methylation of LINE1 (L1) elements, some of which are capable of retrotransposition in human cells, is known to play important roles in transcriptional repression of these retrotransposons. We have previously identified consistent hypomethylation of L1 elements in mouse liver tumors by a genome-wide search technique for aberrant methylations. In this study, we analyzed the methylation status of the L1 elements in human hepatocellular carcinomas (HCCs). METHODS: Nine pairs of an HCC and its surrounding tissue were obtained from clinical cases. Genomic DNA was digested with HpaII, a methylation-sensitive restriction enzyme, and hybridized with a probe derived from the promoter region of the L1 elements. RESULTS: Hypomethylation of the L1 elements was detected in eight of the nine HCCs, but never in the surrounding liver tissues, whether or not liver cirrhosis was present. CONCLUSION: Specific occurrence of the hypomethylation of the L1 elements in the HCCs indicated its diagnostic value for malignancy. The hypomethylation could also lead to increased incidence of retrotransposition and resultant genomic instability in HCCs.

Transcomplementation between different types of respiration-deficient mitochondria with different pathogenic mutant mitochondrial DNAs.

Two cell lines were used for determination of whether interaction occurred between different types of respiration-deficient mitochondria. One was a respiration-deficient rho- cell line having mutant mitochondrial DNA (mtDNA) with a 5,196-base pair deletion including five tRNA genes (tRNAGly, Arg, Ser(AGY), Leu(CUN), His), DeltamtDNA5196, causing Kearns-Sayre syndrome. The other was a respiration-deficient syn- cell line having mutant mtDNA with an A to G substitution at 4,269 in the tRNAIle gene, mtDNA4269, causing fatal cardiomyopathy. The occurrence of mitochondrial interaction was examined by determining whether cybrids constructed by fusion of enucleated rho- cells with syn- cells became respiration competent by exchanging their tRNAs. No cybrids were isolated in selection medium, where only respiration-competent cells could survive, suggesting that no interaction occurred, or that it occurred so slowly that sufficient recovery of mitochondrial respiratory function was not attained by the time of selection. The latter possibility was confirmed by the observations that heteroplasmic cybrids with both mutant mtDNA4269 and DeltamtDNA5196 isolated without selection showed restored mitochondrial respiration activity. This demonstration of transcomplementation between different respiration-deficient mitochondria will help in understanding the relationship between somatic mutant mtDNAs and the roles of such mutations in aging processes.

Isolation and characterization of mitochondrial DNA-less lines from various mammalian cell lines by application of an anticancer drug, ditercalinium.

Since ethidium bromide was not effective in mouse cell lines for isolating mitochondrial DNA (mtDNA)-less cells (rho zero cells), we examined whether an anticancer drug, ditercalinium (DC), which has been shown to exclude mtDNA from mouse cell lines, could be effective in various mouse and human cell lines. We found that after DC treatment rho zero cells could be isolated from all cell lines of mouse or human origin tested. Moreover, these rho zero cells maintained ability to receive exogenously imported mtDNA and allow its replication and gene expression. These observations suggest that DC eliminates mtDNA from mouse and human cells without affecting the property to receive exogenous mtDNA. Therefore, DC could be applicable to cell lines expressing various differentiated phenotypes for studying whether mtDNA plays a significant role in expression of phenotypes by manipulating mtDNA elimination and reintroduction.

Isolation of mitochondrial DNA-less mouse cell lines and their application for trapping mouse synaptosomal mitochondrial DNA with deletion mutations.

For isolation of mouse mtDNA-less (rho0) cell lines, we searched for various antimitochondrial drugs that were expected to decrease the mtDNA content and found that treatment with ditercalinium, an antitumor bis-intercalating agent, was extremely effective for completely excluding mtDNA in all the mouse cell lines we tested. The resulting rho0 mouse cells were successfully used for trapping the mtDNA of living nerve cells into dividing cultured cells by fusion of the rho0 cells with mouse brain synaptosomes, which represent synaptic endings isolated from nerve cells. With neuronal mtDNA obtained, all of the cybrid clones restored mitochondrial translation activity similarly regardless of whether the mtDNA was derived from young or aged mice, thus at least suggesting that defects in mitochondrial genomes are not involved in the age-associated mitochondrial dysfunction observed in the brain of aged mice. Furthermore, we could trap a very small amount of a common 5823-base pair deletion mutant mtDNA (DeltamtDNA5823) that was detectable by polymerase chain reaction in the cybrid clones. As the amount of mutant mtDNA with large scale deletions was expected to increase during prolonged cultivation of the cybrids, these cells should be available for establishment of mice containing the deletion mutant mtDNA.

Identification of inheritance modes of mitochondrial diseases by introduction of pure nuclei from mtDNA-less HeLa cells to patient-derived fibroblasts.

A nuclear genome delivery system was developed to deduce the modes of inheritance of the clinical phenotypes observed in patients with mitochondrial diseases by transfer of pure nuclei from normal cells to fibroblasts from the patients. The problem of possible contamination of the nuclei with a small amount of mtDNA was overcome by using mtDNA-less (rho0) human cells as nuclear donors. In this study, intercellular transfer of pure nuclei was carried out by simple fusion of rho0 HeLa cells with 533 fibroblasts from a patient with a fatal mitochondrial disease, which were deficient in cytochrome c oxidase and succinate dehydrogenase activities. The results showed that the cytochrome c oxidase and succinate dehydrogenase activities were restored by the introduction of pure HeLa nuclei, suggesting that the observed phenotypes of mitochondrial dysfunction were not due to mtDNA mutations but to nuclear, recessive mutations. Thus, our nuclear transfer system is effective for determining whether a mitochondrial or nuclear genome of a patient is responsible for a disease and whether deficiency of mitochondrial enzymes, including enzymes exclusively encoded by nuclear genomes, is transmitted in a nuclear recessive or nuclear dominant way, providing the parents of the patients with valuable information for genetic counseling on the risk of mitochondrial diseases in their next babies.

The interorganellar interaction between distinct human mitochondria with deletion mutant mtDNA from a patient with mitochondrial disease and with HeLa mtDNA.

For the examination of possible intermitochondrial interaction of human mitochondria from different cells, cybrids were constructed by introducing HeLa mitochondria into cells with respiration-deficient (rho-) mitochondria. Respiration deficiency was due to the predominance of mutant mtDNA with a 5,196-base pair deletion including five tRNA genes (DeltamtDNA5196). The HeLa mtDNA and DeltamtDNA5196 encoded chloramphenicol-resistant (CAPr) and chloramphenicol-sensitive (CAPs) 16 S rRNA, respectively. The first evidence for the interaction was that polypeptides exclusively encoded by DeltamtDNA5196 were translated on the introduction of HeLa mitochondria, suggesting supplementation of the missing tRNAs by rho- mitochondria from HeLa mitochondria. Second, the exchange of mitochondrial rRNAs was observed; even in the presence of CAP, CAPs DeltamtDNA5196-specific polypeptides as well as those encoded by CAPr HeLa mtDNA were translated in the cybrids. These phenomena can be explained assuming that the translation in rho- mitochondria was restored by tRNAs and CAPr 16 S rRNA supplied from HeLa mitochondria, unambiguously indicating interorganellar interaction. These observations introduce a new concept of the dynamics of the mitochondrial genetic system and help in understanding the relationship among mtDNA mutations and expression of human mitochondrial diseases and aging.

Mitochondrial DNA is required for regulation of glucose-stimulated insulin secretion in a mouse pancreatic beta cell line, MIN6.

To determine whether mtDNA and mitochondrial respiratory function in pancreatic beta cells are necessary for the phenotypic expression of glucose-stimulated insulin secretion, we used a cultured mouse pancreatic beta cell line, MIN6, and two derivative lines, mtDNA knockout MIN6 (rho0 MIN6) and mtDNA repopulated cybrid MIN6. The MIN6 cells retain the property of glucose-stimulated insulin secretion, but their mtDNA knockout induced the loss of mitochondrial transcription, translation, and respiration activity, without inhibition of transcription of the insulin gene or loss of succinate dehydrogenase activity, indicating that the observed mitochondrial dysfunction in rho0 MIN6 cells was not due to a cytotoxic side effect derived from the mtDNA knockout. Moreover, the mtDNA depletion also inhibited both the glucose-stimulated increase in the intracellular free Ca2+ content and the elevation of insulin secretion. The possibility of the involvement of nuclear genome-encoded factors in this process was excluded by the observation that the missing sensitivity to extracellular glucose stimulation in rho0 MIN6 cells was restored reversibly by repopulation with foreign mtDNA and isolating cybrid MIN6 clones. Therefore, these findings provide unambiguous evidence for the involvement of the mitochondrial dysfunction induced by mtDNA impairment in developing pathogeneses of some forms of diabetes mellitus.

Mutant mtDNA at 1555 A to G in 12S rRNA gene and hypersusceptibility of mitochondrial translation to streptomycin can be co-transferred to rho 0 HeLa cells.

Human skin fibroblast line 95-119, which had been isolated from the mother of a Japanese patient with aminoglycoside-induced deafness and a 1555 A to G mutation at 12S rRNA gene in mitochondrial DNA (mtDNA), was used to investigate the relationship between the 1555 mtDNA mutation and its pathogenicity. By the intercellular transfer of mtDNA with or without the 1555 mutation to mtDNA-less (rho 0) HeLa cells, we isolated cybrid clones and found that the mitochondrial translation in a cybrid clone repopulated with the homoplasmic 1555 mutation showed the highest susceptibility to streptomycin. These observations suggest that the genotype of the mutant mtDNA and the phenotype of hypersusceptibility to streptomycin observed in 95-119 fibroblasts were co-transferred simultaneously to rho 0 HeLa cells, supporting the idea that the homoplasmic 1555 mtDNA mutation is involved in the pathogenesis leading to aminoglycoside-induced hearing loss.

Age-associated changes of mitochondrial translation and respiratory function in mouse brain.

We examined age-associated changes of respiratory enzyme activities and protein synthesis in mitochondria isolated from mouse brain with high oxidative activities. Cytochrome c oxidase activity increased unexpectedly with aging, while the mitochondrial translational activities showed two phases of alterations: they increased progressively up to 21 weeks after birth followed by a gradual decrease with aging. Results showed that these changes were not due to the change in mitochondrial DNA (mtDNA) copy number or the accumulation of deletion mutations in mtDNA. These observations suggest that the common feature of age-associated changes in both human and mouse mitochondrial functions is limited to the decrease in mitochondrial translational activity. Therefore, mouse brain can be used as a model to understand the relationships between aging and mitochondrial function by examining the cause of decrease in mitochondrial translation activity.

Functional and morphological abnormalities of mitochondria in human cells containing mitochondrial DNA with pathogenic point mutations in tRNA genes.

mtDNA with a point mutation in the tRNA(Ile) gene at nucleotide position 4269 found in a patient with fatal cardiomyopathy and mtDNA with a point mutation in the tRNA(Arg) gene at 10410 found in a patient with Alpers disease were transferred cytoplasmically to rho zero HeLa cells (HeLa cells lacking mtDNA) to determine whether these novel mtDNA mutations in the tRNA genes are responsible for the defects in mitochondrial respiration function observed in these diseases. Cybrid clones (clones of rho zero HeLa cells with mtDNA from the patients) were isolated, and respiratory function and morphology of the mitochondria of the cybrid clones containing wild-type mtDNA and mutant mtDNA predominantly were compared. The results showed that accumulation of mutant mtDNA at 4269 alone without defects in the nuclear genome was sufficient to produce a disease phenotype, while mutant mtDNA at 10410 was not related to pathogenesis and reflected one of the rare polymorphic sites of human mtDNA. Moreover, we found that mitochondria in living cells were significantly swollen only when they contained predominantly the pathogenic mutant mtDNA, suggesting that the functional abnormality of mitochondria induced by pathogenic mtDNA mutations in tRNA genes is always associated with their swollen structure.

Nuclear but not mitochondrial genome involvement in human age-related mitochondrial dysfunction. Functional integrity of mitochondrial DNA from aged subjects.

The role of mtDNA and nuclear genome in human aging was examined by their intercellular transfer using skin fibroblasts and mtDNA-less HeLa cells (rho o-HeLa cells). We found in vivo age-related reductions in the activity of cytochrome c oxidase in human skin fibroblasts obtained from 16 donors of various ages (0-97 years). The abnormality in mitochondria of the aged donors was not attributable to either decrease in the copy number of mtDNA molecules or increase in the copy number of deletion mutant mtDNA molecules, but to significant decrease in overall polypeptide synthesis in the mitochondria. However, intercellular mtDNA transfer experiments showed that fibroblast mtDNA from elderly donors is functionally intact. By contrast, intercellular transfer of HeLa nuclei to fibroblasts from aged donors restored cytochrome c oxidase activity, suggesting that the age-related phenotype was nuclear recessive. However, during subsequent cultivation of these hybrids, the activity gradually reduced again, associated with gradual chromosome loss. These observations support the idea that accumulation of nuclear recessive somatic mutations, but not mtDNA mutations, is responsible for the in vivo age-related mitochondrial dysfunction observed in human skin fibroblasts.

Detection and characterization of muscle-specific nuclear proteins.

To identify nuclear proteins related to muscle tissue specificity, we tried to prepare antibodies recognizing muscle-specific nuclear proteins. Taking advantage of the autoimmunity of some nuclear proteins, we prepared an antiserum against chick muscle nuclear proteins by injecting protein components of the nuclei isolated from chick breast muscles into breast muscle. Three proteins, named p30, p32, and p37, were detected with the antiserum in a two-dimensional SDS-PAGE pattern of the isolated nuclei. P30 and p32 were not detected in the nuclei of liver, brain, cardiac muscle, or slow type skeletal muscle (anterior latissimus dorsi). They were detected in those of fast type skeletal muscle (pectoralis major and semitendinosus) and smooth muscle (gizzard) at all developmental stages examined. On serial fractionation of muscle cell nuclei, they were detected in a fraction obtained after DNase I treatment of the sample, suggesting that the proteins weakly bind to chromatin. A homology search of amino acid sequences showed that there is no known protein similar to p32.