Biomedical subjects

M Oshimura

Publications and source records attributed to M Oshimura.

At least 55 records · Page 3Linked to original sources

PubMed2000

Construction of human-rodent hybrid cells containing single transferable fragments of human chromosome 10p.

The introduction of chromosome 10p into human glioblastoma or prostate cancer cells has been demonstrated to suppress their malignant phenotype, suggesting the presence of glioma or prostate tumor suppressor genes on 10p. As a resource for the fine mapping of these genes, a series of human-rodent hybrid cell lines containing single transferable fragments (STFs) of 10p were constructed. Normal chromosome 10 tagged with a neomycin-resistance gene on its short arm was fragmented by gamma-irradiation of 5-10krad, transferred into mouse L cells or Chinese hamster ovary cells by microcell-mediated chromosome transfer (MMCT), and then selected against G418. Thirty-three independent rodent-human hybrids carrying various-sized STFs were obtained. Polymerase chain reaction (PCR)-based genotyping revealed that these STFs contained the whole, or portions, of a 43-cM region on 10p14-pter and could be defined by 19 sequence-tagged-site (STS) markers. Using this panel of hybrids as donors for further MMCT, genes on the refined fragments could be transferred into other cells. This hybrid panel would therefore be a useful resource for the fine mapping of the genes on 10p14-pter to segments of about 2.4 cM by functional complementation.

Animals↗

PubMed2000

Human monochromosome hybrid cell panel characterized by FISH in the JCRB/HSRRB.

The human monochromosome hybrid cell panel in the Japanese Collection of Research Bioresources (JCRB) consists of 23 mouse cell clones, each containing a different human chromosome (the Y chromosome is not yet included). The panel is currently distributed by the Human Science Research Resources Bank (HSRRB) in Osaka. In order to determine the state of the human chromosomes and to supply the information to investigators, we characterized the cells by fluorescence in-situ hybridization (FISH) with corresponding human chromosome-specific painting probes, and, in part, by reverse FISH with the hybrid total DNA hybridized onto human metaphase spreads. Here, we report the frequency of intact human chromosomes maintained in each hybrid and the retained subregions of corresponding human chromosomes with relative frequencies estimated by fluorescent intensity. We used specific painted patterns to classify each hybrid into tentative types with their frequencies showing the nature of each hybrid and the state of rearrangements. This characterization will provide valuable information to investigators using the panel.

Animals↗

PubMed2000

Stability of transferred human chromosome fragments in cultured cells and in mice.

Chromosome fragments represent feasible gene delivery vectors with the use of microcell-mediated chromosome transfer. To test a prerequisite for a gene delivery vector, we examined the stability of human chromosome fragments (hCFs) in cultured cells and in trans-chromosomic (Tc) mice. Fragments of human chromosomes 2 (hCF(2-W23)), 11 (hCF-11) and 14 (hCF(SC20)) tagged with neo were introduced into the TT2F mouse ES cells, and retention of the hCFs was examined by FISH during long-term culture without selection. In contrast to the gradual loss of hCF(2-W23) and hCF-11, hCF(SC20) remained stable over 70 population doublings in the ES cells. The hCF(SC20) was also stable in cultured human tumor cells and chicken DT40 cells. We have previously generated chimeric mice using the ES cells harboring the hCF(2-W23) or hCF(SC20), followed by production of Tc mice. Although both the hCF(2-W23) and hCF(SC20) persisted in cells of Tc mice as an additional chromosome and were transmitted to offspring, the hCF(SC20) was more stable than the hCF(2-W23) in F1 and F2 mice. The present study shows that the stability of hCFs in Tc mice differs with tissue types and with genetic background used for successive breedings. Thus, the hCF(SC20), which was relatively stable in both mouse and human cells, may be a promising candidate for development as a gene delivery vector.

Animals↗

PubMed2000

Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts.

For introducing regions of human chromosomes greater than a megabase into cells or animals, we have developed a chromosome-cloning system in which defined regions of human chromosomes can be cloned into a stable human minichromosome vector in homologous recombination-proficient chicken DT40 cells. The stable minichromosome vector allowed a 10 Mb-sized region of the mitotically unstable human chromosome 22 to be stably maintained in mouse embryonic stem (ES) cells, and in mice. Furthermore, we demonstrated functional expression of human genes from the HAC in mice. This study describes a stable cloning and expression system for greater than megabase-sized regions of human chromosomes.

Animals↗

PubMed2000

Telomerase activity significantly correlates with chromosome alterations, cell differentiation, and proliferation in lung adenocarcinomas.

Telomerase activity was examined by the telomeric repeat amplification protocol assay in 25 cases of lung adenocarcinoma, in relation to cancer cell differentiation, proliferation, and chromosome alterations. Telomerase activity, chromosome alterations, and cell proliferation assessed by Ki-67 labeling were significantly lower (P < .001 to .05) in well-differentiated (10 cases) than in moderately differentiated (8 cases) or poorly differentiated (7 cases) lesions. Telomerase activity by semiquantitative analysis with scoring of 0 to 3 was significantly correlated with similarly graded chromosome alterations (P < .05) and Ki-67 labeling indices (P < .002). Telomerase activity and chromosome alteration (T-C) indices generated by multiplication of telomerase activity and chromosome alteration scores also showed a significant correlation with cell differentiation. The Clara cell subtype, confirmed by electron microscopic analysis, significantly predominated in the well-differentiated group, showing a low grade of telomerase activity and chromosome alterations and low Ki-67 labeling indices, suggesting clinical relevance. No significant association of telomerase activity was found with p53 protein accumulation or Bcl-2 protein expression. The good correlation of telomerase activity with chromosome alterations, cell differentiation, and Ki-67 labeling indices suggests that this parameter might have potential application in estimation of prognosis.

Adenocarcinoma↗

PubMed2000

Telomere shortening with aging in human liver.

Progressive telomere shortening with aging was studied in the normal liver tissue of 94 human subjects aged between 0 and 101 years old to determine the rate of telomere loss in 1 year. Telomere length demonstrated accelerated shortening with reduction of 55 base pairs (bp) per year. The mean telomere length in five neonates was 12.9 +/- 2.6 kilobase pairs (kbp), and that in one centenarian was 8.3 kbp. Mean telomere lengths by age group were 13.2 +/- 2.0 kbp (< or = 8 years; 10 subjects), 7.8 +/- 1.9 kbp (40-79 years; 29 subjects), and 7.5 +/- 2.0 kbp (> or = 80 years; 53 subjects), with reduction thus appearing to show slowing on the attainment of middle age. The difference of mean telomere lengths for two groups with or without advanced malignancies of other than liver origin was not significant in the older two groups. Despite the slow turnover of liver tissue, the overall reduction rate of telomere length decrease in 1 year was almost the same as that of digestive tract mucosa, with its very rapid renewal.

Adolescent↗

PubMed2000

Increased frequencies of gene and chromosome mutations after X-irradiation in mouse embryonal carcinoma cells transfected with the bcl-2 gene.

Preimplantation stage mouse embryos are known to be highly sensitive to the killing effect of DNA-damaging agents such as radiation. Interestingly, however, this stage of development is well protected from radiation induction of malformation and carcinogenesis in postnatal life. In recent years, it has become clear that the stem cells of preimplantation stage embryos undergo extensive apoptosis after DNA damage. It has been postulated that this apoptosis is likely to be responsible for the resistance to malformation, by excluding cells carrying deleterious DNA damage. We have tested the possible role of apoptosis in elimination of gene and chromosome mutations in undifferentiated mouse embryonal carcinoma cell line, F9, transfected with human bcl-2 cDNA. The colony radiosensitivity of F9 cells was not affected by overexpression of the bcl-2 gene, but the apoptotic cell death was suppressed, as examined by DNA ladder assay and Hoechst staining. This suppression was accompanied by an increase in the frequencies of hprt mutation and micronucleus formation after X-irradiation. These results support the idea that maintenance of genomic integrity during early development is likely to be executed by apoptotic elimination of cells at risk.

Animals↗

PubMed2000

Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified telomeric DNA.

Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER(-/-) cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicates cis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.

Animals↗

PubMed2000

Epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events involving imprinted genes at chromosome 11p15.5. Thus, germline mutations in CDKN1C, uniparental disomy (UPD), and loss of imprinting of IGF2 and other imprinted genes have been implicated. Many familial BWS cases have germline CDKN1C mutations. However, most BWS cases are sporadic and UPD or putative imprinting errors predominate in this group. We have identified previously a subgroup of sporadic cases with loss of imprinting (LOI) of IGF2 and epigenetic silencing of H19 proposed to be caused by a defect in a distal 11p15.5 imprinting control element (designated BWSIC1). However, many sporadic BWS patients show biallelic IGF2 expression in the presence of normal H19 methylation and expression patterns. This and other evidence suggested the existence of a further imprinting control element (BWSIC2) at 11p15. 5. Recently, we showed that a subgroup of BWS patients have loss of methylation (LOM) at a differentially methylated region (KvDMR1) within the KCNQ1 gene centromeric to the IGF2 and H19 genes. We have now analysed a large series of sporadic cases to define the frequency and phenotypic correlates of epigenetic abnormalities in BWS. LOM at KvDMR1 was detected by Southern analysis or a novel PCR based method in 35 of 69 (51%) sporadic BWS without UPD. LOM at KvDMR1 was often, but not invariably associated with LOI of IGF2. KvDMR1 LOM was not detected in BWS patients with putative BWSIC1 defects and cases with KvDMR1 LOM (that is, putative BWSIC2 defects) invariably had a normal H19 methylation pattern. The incidence of exomphalos in putative BWSIC2 defect patients was not significantly different from that in patients with germline CDKN1C mutations (20/29 and 13/15 respectively), but was significantly greater than that in patients with putative BWSIC1 defects (0/5, p=0.007) and UPD (0/22, p<0.0001). These findings are consistent with the hypothesis that LOM of KvDMR1 (BWSIC2 defect) results in epigenetic silencing of CDKN1C and variable LOI of IGF2. BWS patients with embryonal tumours have UPD or a BWSIC1 defect but not LOM of KvDMR1. This study has further shown how (1) variations in phenotypic expression of BWS may be linked to specific molecular subgroups and (2) molecular analysis of BWS can provide insights into mechanisms of imprinting regulation.

Beckwith-Wiedemann Syndrome↗

PubMed2000

The effect of caffeine on p53-dependent radioresponses in undifferentiated mouse embryonal carcinoma cells after X-ray and UV-irradiations.

The effect of caffeine was studied on the radioresponses of undifferentiated mouse embryonal carcinoma cells (EC cells) with or without the functional p53. The radioresponses studied included radiosensitivity, the activation of p53, apoptosis with characteristic DNA ladder formation and cell cycle progression. An undifferentiated mouse EC cell line, ECA2, and a newly established p53-deficient EC cell line, p53 delta, were used in the present study. The status of the p53 gene did not significantly affect the colony survivals of undifferentiated EC cells to X-rays and UV. Although a post-irradiation treatment with caffeine sensitized both lines to X-rays marginally, the sensitization was prominent for UV regardless of the p53 status of the cells. The activation of a p53 responsible lacZ reporter construct was observed in stably transfected ECA2 cells after X-ray and UV irradiations. Caffeine suppressed the X-ray induced activation of the lacZ reporter, while it drastically enhanced the activation after UV irradiation. X-rays and UV readily triggered the apoptosis of ECA2 cells with the characteristic DNA ladder. Although UV-induced DNA ladder formation was enhanced by caffeine, that induced by X-rays was unaffected. Therefore, the effects of caffeine on the p53-dependent radioresponses were found to be agent specific: suppression for the X-ray induced and augmentation for the UV induced. In contrast to p53-proficient ECA2 cells, smear-like DNA degradation was observed for irradiated p53 delta cells, suggesting the presence of a mode of cell death without DNA ladder formation. UV induction of the smear-like DNA degradation was enhanced in the presence of caffeine. Regardless of the state of the p53 gene, G1/S arrest was not observed in X-ray and UV irradiated EC cells. X-ray induced G2/M arrest in both lines, which was abrogated by caffeine, while G2/M arrest after UV was unaffected by a caffeine treatment. These results indicate that the radioresponses of undifferentiated EC cells differ considerably from those of somatic cells, and that these radioresponses were modulated by a post-irradiation treatment with caffeine.

Animals↗

PubMed2000

Multipoint imprinting analysis in sporadic colorectal cancers with and without microsatellite instability.

Disrupted imprinting is implicated in certain tumorigenesis. Since aberrant methylation has been described for a majority of microsatellite instability (MSI)-positive sporadic colorectal cancers, we have investigated alteration to the imprinting in 55 sporadic colorectal cancers with or without MSI. Loss of imprinting (LOI) of IGF2 and PEG1/MEST was observed in 42% and 35% of informative cancers, respectively. H19 expression was not detected in 24% of informative cancers. SNRPN and NDN retained monoallelic expression in all the cancers examined. These findings indicate no simultaneous disruption of the imprinted genes. LOI of IGF2 and PEG1/MEST was also observed in colorectal mucosa from almost all the patients with LOI in tumor tissue. Moreover, MSI-positive colorectal cancers exhibit LOI of IGF2 with a high frequency compared to MSI-negative cancers (P=0.013). These observations, consistent with a previous report, establish an association between LOI of IGF2 and MSI in colorectal cancers and provide insight into susceptibility of tumor development.

Alleles↗

PubMed2000

The expansion of the CAG repeat in exon 1 of the human androgen receptor gene is associated with uterine endometrial carcinoma.

Uterine endometrial carcinoma is common among women. Several reports indicated that this cancer is influenced by androgens mediated through their receptors. The human androgen receptor gene contains a polymorphic CAG repeat in the coding region of exon 1. Other studies suggested a possible association between the CAG repeat of this gene and the development of several cancers. DNA samples isolated from 29 patients with sporadic endometrial cancer were analyzed for allelic changes in 12 highly polymorphic microsatellite loci on nine chromosomes, containing CAG repeat in the exon 1 of the androgen receptor gene. A significantly high rate of allelic change in the CAG repeat was observed (51.7%) in these patients, although the frequencies of additional loci were similar to those reported by others (0-22.2%). The changed alleles of the tumor tissue were always longer than that of normal tissue except in only one case. Since only one allele on the X chromosome is commonly active in female cells, a differential methylation assay was carried out by using genomic DNA cut with HpaII. In 14 of 15 cases, we found that the activated allele was longer in these samples from tumor tissues than those from normal tissue; in the remaining case, the length of this repeat was unchanged. The expression assays were done by using poly(A+) RNA from tumor and normal uterine tissues, revealing that an activated allele in these tumor tissues was longer than that in the normal tissues in all the cases examined. These findings suggest that expansions of the CAG repeat in the androgen receptor gene may play an important role in the carcinogenesis of uterine endometrial cells.

Alleles↗

PubMed2000

Mapping of metastasis suppressor genes for prostate cancer by microcell-mediated chromosome transfer.

AIM: To identify the metastasis suppressor genes for prostate cancer. METHODS: A copy of human chromosomes was introduced into the highly metastatic Dunning R-3327 rat prostate cancer cells by the use of microcell-mediated chromosome transfer. Relationships between the size of human chromosomes introduced into microcell hybrid clones and the number of lung metastases produced by the clones were analyzed to determine which part of human chromosomes contained the metastasis suppressor gene(s) for prostate cancer. To determine portions of human chromosomes introduced, G-banding chromosomal analysis, fluorescence in situ hybridization analysis, and polymerase chain reaction analysis were performed. RESULTS: Each of microcell hybrid clones containing human chromosomes 7, 8, 10, 11, 12, or 17 showed decreased ability to metastasize to the lung without any loss of tumorigenicity. This demonstrates that these human chromosomes contain metastasis suppressor genes for prostate cancer. Spontaneous deletion of portions of human chromosomes was observed in the human chromosome 7, 10, 11, 12, and 17 studies. In the human chromosome 8 study, irradiated microcell-mediated chromosome transfer was performed to enrich chromosomal arm deletions of human chromosome 8. Molecular and cytogenetic analyses of microcell hybrid clones demonstrated that metastasis suppressor genes on human chromosomes were located on 7q21-22, 7q31.2-32, 8p21-12, 10q11-22, 11p13-11.2, 12p11-q13, 12q24-ter, and 17pter-q23. KAI1 and MKK4/SEKI were identified as metastasis suppressor genes from 11p11.2 and 17p12, respectively. CONCLUSION: This assay system is useful to identify metastasis suppressor gene (s) for prostate cancer.

Animals↗

PubMed1999

Telomerase activity correlates with histo-pathological factors in uterine endometrial carcinoma.

Telomerase activity has been implicated in the progression of various human tumors. Our aim was to evaluate telomerase activity and to compare it with histo-pathological factors in uterine endometrial carcinoma, to look for possible correlations. Telomerase activity was measured by dilution analysis using a PCR-based telomeric repeat amplification method and detected in 31 of 35 primary endometrial carcinoma tumor specimens. High telomerase activity, detected after 100-fold dilution of extracts, was identified in 15 specimens. There was no significant correlation between the positive telomerase activity and tumor surgical stage or histo-pathological factors. However, high telomerase activity was significantly correlated with advanced surgical stage and with pelvic lymph node metastasis. Our findings suggest that an increase in telomerase activity may be associated with tumor progression and that its level may have a prognostic value in endometrial carcinoma.

Adult↗

PubMed1999

Mouse A9 cells containing single human chromosomes for analysis of genomic imprinting.

To develop an systematic in vitro approach for the study of genomic imprinting, we generated a new library of human/mouse A9 monochromosomal hybrids. We used whole cell fusion and microcell-mediated chromosome transfer to generate A9 hybrids containing a single, intact, bsr-tagged human chromosome derived from primary fibroblasts. A9 hybrids were identified that contained either human chromosome 1, 2, 4, 5, 7, 8, 10, 11, 15, 18, 20, or X. The parental origin of these chromosomes was determined by polymorphic analysis using microsatellite markers, and matched hybrids containing maternal and paternal chromosomes were identified for chromosomes 5, 10, 11 and 15. The imprinted gene KVLQT1 on human chromosome 11p15.5 was expressed exclusively from the maternal chromosome in A9 hybrids, and the parental-origin-specific expression patterns of several other imprinted genes were also maintained. This library of human monochromosomal hybrids is a valuable resource for the mapping and cloning of human genes and is a novel in vitro system for the screening of imprinted genes and for their functional analysis.

Animals↗

PubMed1999

The XRCC2 and XRCC3 repair genes are required for chromosome stability in mammalian cells.

The irs1 and irs1SF hamster cell lines are mutated for the XRCC2 and XRCC3 genes, respectively. Both show heightened sensitivity to ionizing radiation and particularly to the DNA cross-linking chemical mitomycin C (MMC). Frequencies of spontaneous chromosomal aberration have previously been reported to be higher in these two cell lines than in parental, wild-type cell lines. Microcell-mediated chromosome transfer was used to introduce complementing or non-complementing human chromosomes into each cell line. irs1 cells received human chromosome 7 (which contains the human XRCC2 gene) or, as a control, human chromosome 4. irs1SF cells received human chromosome 14 (which contains the XRCC3 gene) or human chromosome 7. For each set of hybrid cell lines, clones carrying the complementing human chromosome recovered MMC resistance to near-wild-type levels, while control clones carrying noncomplementing chromosomes remained sensitive to MMC. Fluorescence in situ hybridization with a human-specific probe revealed that the human chromosome in complemented clones remained intact in almost all cells even after extended passage. However, the human chromosome in noncomplemented clones frequently underwent chromosome rearrangements including breaks, deletions, and translocations. Chromosome aberrations accumulated slowly in the noncomplemented clones over subsequent passages, with some particular deletions and unbalanced translocations persistently transmitted throughout individual subclones. Our results indicate that the XRCC2 and XRCC3 genes, which are now considered members of the RAD51 gene family, play essential roles in maintaining chromosome stability during cell division. This may reflect roles in DNA repair, possibly via homologous recombination.

Animals↗

PubMed1999

Loss of imprinting of a paternally expressed transcript, with antisense orientation to KVLQT1, occurs frequently in Beckwith-Wiedemann syndrome and is independent of insulin-like growth factor II imprinting.

Genomic imprinting plays a fundamental role in cancer and some hereditary diseases, including Beckwith-Wiedemann syndrome (BWS), a disorder of prenatal overgrowth and predisposition to embryonal malignancies such as Wilms tumor. We have previously shown that the KVLQT1 gene on chromosomal band 11p15 is imprinted, with expression of the maternal allele, and that the maternal allele is disrupted in rare BWS patients with balanced germ-line chromosomal rearrangements. We now show that an antisense orientation transcript within KVLQT1, termed LIT1 (long QT intronic transcript 1) is expressed normally from the paternal allele, from which KVLQT1 transcription is silent, and that in the majority of patients with BWS, LIT1 is abnormally expressed from both the paternal and maternal alleles. Eight of sixteen informative BWS patients (50%) showed biallelic expression, i.e., loss of imprinting (LOI) of LIT1. Similarly, 21 of 36 (58%) BWS patients showed loss of maternal allele-specific methylation of a CpG island upstream of LIT1. Surprisingly, LOI of LIT1 was not linked to LOI of insulin-like growth factor II (IGF2), which was found in 2 of 10 (20%) BWS patients, even though LOI of IGF2 occurs frequently in Wilms and other tumors, and in some patients with BWS. Thus, LOI of LIT1 is the most common genetic alteration in BWS. We propose that 11p15 harbors two imprinted gene domains-a more centromeric domain including KVLQT1 and p57(KIP2), alterations in which are more common in BWS, and a more telomeric domain including IGF2, alterations in which are more common in cancer.

Beckwith-Wiedemann Syndrome↗

PubMed1999

Metastasis suppressor gene(s) for rat prostate cancer on the long arm of human chromosome 7.

Allelotype analyses of human prostate cancer indicate that allelic losses on human chromosome arms 7q, 8p, 10q, 13q, 16q, 17q, and 18q are observed frequently. For the study of the possible biological significance of the frequently observed deletions on chromosome arm 7q in human prostate cancer, human chromosome 7 was introduced into highly metastatic rat prostate cancer cells by use of a microcell-mediated chromosome transfer technique. The introduction of human chromosome 7 resulted in the suppression of metastatic ability of the microcell hybrids, whereas no suppression of tumorigenicity was observed. To identify the portion of chromosome 7 containing the metastasis-suppressive function gene, the derivative chromosome 7 that was generated with the initial transfer was retransferred into rat prostate cancer cells. Human chromosome 7-containing rat prostate cancer cells could be used as the donor cells, because rodent cells produced a sufficient number of microcells with colchicine treatment. Cytogenetic and molecular analyses of these clones demonstrated that loss of segments on 7q was related to the reexpression of the metastatic phenotype. These results show that human 7q contains a metastasis suppressor gene or genes for rat prostate cancer. The findings also suggest that this gene may play an important role in the progression of human prostate cancer.

Animals↗