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M Oshimura

Publications and source records attributed to M Oshimura.

At least 73 records · Page 4Linked to original sources
PubMed1999

Telomerase-independent senescence of human immortal cells induced by microcell-mediated chromosome transfer.

Maintenance of telomeres, commonly through expression of telomerase activity, is necessary but may not be sufficient for human cells to escape from the cellular senescence program and become immortal. We report here that human tumor cells could undergo cellular senescence in the presence of telomerase activity when a specific normal human chromosome was introduced via microcell-mediated chromosome transfer. The cell models studied include SiHa (uterine cervical carcinoma cells expressing E6 and E7 oncoproteins of human papillomavirus type 16) with a transferred chromosome 2, CC1 (choriocarcinoma cells expressing an amino-terminally truncated p53 protein) with a transferred chromosome 7, and JTC-32 (bladder carcinoma cells) with a transferred chromosome 11. The microcell hybrids with the indicated chromosomes ceased to divide after five to 10 population doublings and showed senescence-associated beta-galactosidase activity but still expressed the genes encoding three components of human telomerase, consistent with the retention of telomerase activity. These results are evidence for barriers to human cell immortalization, which involve activation of unidentified senescence-inducing genes that function independently of inactivation of telomerase.

Cell Line, Transformed↗
PubMed1999

Clinical usefulness of telomerase activity and telomere length in the preoperative diagnosis of gastric and colorectal cancer.

It has been reported that telomerase activity and telomeric reduction can be detected in many human cancers. Although it is well known that telomerase activity and telomere length have important implications for cancer biology, their clinical usefulness in the preoperative diagnosis of gastric and colorectal cancer has not been elucidated. Therefore, we examined telomerase activity and telomere length in gastric and colorectal cancer using tissue samples obtained by fiberscopy. Telomerase activity was measured by a telomeric repeat amplification protocol (TRAP). Although telomerase activity was detected in 1/12 (8%) cases of gastric polyp and in 2/9 (22%) cases of colorectal polyp, its positivity in gastric cancer and colorectal cancer was 7/10 (70%) and 21/26 (81%; P<0.0003 and P<0.0001, respectively). Telomere length was analyzed by Southern blotting, and telomeric reduction in gastric cancer was significantly greater than that in gastric polyp (P<0.0003). However, there was no telomeric reduction between colorectal cancer and colorectal polyp. The results of the present study indicate that determination of telomerase activity and telomere length may serve as a useful method for preoperative diagnosis of gastric and colorectal cancer.

Adult↗
PubMed1999

LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids.

Mammalian imprinted genes are frequently arranged in clusters on particular chromosomes. The imprinting cluster on human chromosome 11p15 is associated with Beckwith-Wiedemann syndrome (BWS) and a variety of human cancers. To clarify the genomic organization of the imprinted cluster, an extensive screen for differentially expressed transcripts in the 11p15 region was performed using monochromosomal hybrids with a paternal or maternal human chromosome 11. Here we describe an imprinted antisense transcript identified within the KvLQT1 locus, which is associated with multiple balanced chromosomal rearrangements in BWS and an additional breakpoint in embryonal rhabdoid tumors. The transcript, called LIT1 (long QT intronic transcript 1), was expressed preferentially from the paternal allele and produced in most human tissues. Methylation analysis revealed that an intronic CpG island was specifically methylated on the silent maternal allele and that four of 13 BWS patients showed complete loss of maternal methylation at the CpG island, suggesting that antisense regulation is involved in the development of human disease. In addition, we found that eight of eight Wilms' tumors exhibited normal imprinting of LIT1 and five of five tumors displayed normal differential methylation at the intronic CpG island. This contrasts with five of six tumors showing loss of imprinting of IGF2. We conclude that the imprinted gene domain at the KvLQT1 locus is discordantly regulated in cancer from the imprinted domain at the IGF2 locus. Thus, this positional approach using human monochromosomal hybrids could contribute to the efficient identification of imprinted loci in humans.

Animals↗
PubMed1998

Failure to complement abnormal phenotypes of simian virus 40-transformed Werner syndrome cells by introduction of a normal human chromosome 8.

The Werner syndrome (WS) gene (WRN) was isolated by positional cloning, based on mapping to chromosome 8p12, and the WRN protein was recently shown to encode an active helicase. To examine functional complementation of WS phenotypes by expression of the WRN gene, we introduced a normal human chromosome 8 into a SV40-transformed WS fibroblastoid cell line (WS780) by microcell fusion and studied several cellular phenotypes associated previously with WS cell lines, including cell growth rate, 4-nitroquinoline-1-oxide (4NQO) sensitivity, and spontaneous mutation rate and type of mutation at the hypoxanthine-guanine phosphoribosyl transferase locus. The WRN gene was expressed in two of three microcell hybrids introduced with chromosome 8. We failed to observe a difference between normal and WS cell lines in terms of growth rates and spontaneous mutation rates. However, we found that the WS cell line was highly sensitive to 4NQO-induced cytotoxicity and showed an unusually high proportion of deletion mutations at the hypoxanthine-guanine phosphoribosyl transferase locus as compared to a control cell line, as shown previously. Here, we report that these phenotypes were not corrected by introduction of the WRN gene, although the WRN protein was expressed. Our results suggest that the hypersensitivity to 4NQO and the extensive deletion mutations observed in the WS cell line are caused by a defect that is secondary to the WRN gene mutation, possibly a repair gene defect that controls the phenotypes of hypersensitivity to carcinogen(s) and/or the extensive deletion mutations.

4-Nitroquinoline-1-oxide↗
PubMed1998

Efficient modification of a human chromosome by telomere-directed truncation in high homologous recombination-proficient chicken DT40 cells.

Truncation of human chromosomes at desired sites by homologous recombination techniques enables functional and structural analyses of human chromosomes and development of human artificial chromosomes. However, this targeted truncation has been inefficient. We describe here an efficient method for targeted truncation in the chicken DT40 cells with a high homologous recombination rate. The human chromosome 22 was transferred into DT40 cells, where human telomeric repeat (TTAGGG)n was targeted to the LIF locus on the chromosome. Molecular and cytogenetic analyses showed that the predicted truncation at the LIF locus occurred in all of the targeted clones.

Animals↗
PubMed1998

XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation.

DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7 / SCID ( Prkdc ) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.

Animals↗
PubMed1998

Evidence for a putative telomerase repressor gene in the 3p14.2-p21.1 region.

Telomeres, which are the repeated sequences located on both ends of chromosomes in eukaryotes, are known to shorten with each cell division, and their eventual loss is thought to result in cellular senescence. Unlike normal somatic cells, most tumor cells show activation of telomerase, a ribonucleoprotein enzyme that stably maintains telomere length by addition of the sequences of TTAGGG repeats to telomeres. The KC12 cell line derived from a renal cell carcinoma in a patient with von Hippel-Lindau disease showed telomerase activity and loss of heterozygosity on the short arm of chromosome 3. Introduction of a normal human chromosome 3 into KC12 cells by microcell fusion induced cellular senescence, accompanied by suppression of telomerase activity and shortening of telomere length. Microcell hybrids that escaped from cellular senescence maintained telomere length and telomerase activity similar to those of the parental KC12 cells. We previously showed a similar suppression of telomerase activity by introduction of chromosome 3 into another renal cell carcinoma cell line, RCC23. The putative telomerase repressor gene was mapped to chromosome region 3p14.2-p21.1 by deletion mapping of KC12 + chromosome 3 revertants that escaped from cellular senescence and by transfer of subchromosomal fragments of chromosome 3 into RCC23 cells.

Carcinoma, Renal Cell↗
PubMed1998

A repressor function for telomerase activity in telomerase-negative immortal cells.

Human telomerase, a ribonucleoprotein that adds TTAGGG repeats onto telomeres and compensates for their shortening, is repressed in most normal human somatic cells. Human somatic cells are considered to have a limited proliferation capacity because of the telomere shortening. Although immortalization of somatic cells is often associated with telomerase reactivation, there are some immortal cells in which telomerase activity is undetectable. In these cells, telomeres may be maintained by an unknown mechanism other than telomerase reactivation. To examine the genetic regulation of telomerase activity, we constructed hybrids between immortal cells with (HepG2) and without (KMST6) telomerase activity. These two cell lines had relatively short and long telomeres, respectively. The hybrid cells continued to proliferate without detectable telomerase activity even after 100 population doublings. Telomerase-positive subpopulations occasionally appeared after serial passages. Southern blot analysis revealed that the hybrids had long terminal restriction fragments similar to that of KMST6, regardless of telomerase activity, and fluorescence in situ hybridization with a telomeric probe showed high-intensity hybridization signals on telomeres, indicating relatively long telomeric repeats. These results suggest that the telomerase-negative immortal cells contain a gene or genes functioning as a telomerase repressor and maintain telomere length by a dominant mechanism other than telomerase reactivation.

Cells, Cultured↗
PubMed1998

Mapping a novel cellular-senescence gene to human chromosome 2q37 by irradiation microcell-mediated chromosome transfer.

To identify the subchromosomal region that carries the cellular-senescence-restoring program of the human cervical carcinoma cell line SiHa, we constructed by irradiation microcell-mediated chromosome transfer a library of mouse A9 cells containing various fragments of human chromosome 2 tagged with pSV2neo in 2p11-p12. Eighty-seven clones were isolated and screened for the presence of human sequences by inter-Alu and inter-L1 polymerase chain reaction (PCR), and six clones exhibiting PCR-laddering patterns that differed from those of the A9 cells containing an intact chromosome 2 were examined further. Chromosome analysis and fluorescence in situ hybridization (FISH) using human-specific repetitive sequences revealed that four of these clones contained single subchromosomal transferable fragments (STFs). Southern blot hybridization of 14 cosmid markers revealed that the STFs in A9 cells were derived from human chromosome 2. These STFs were transferred into SiHa cells by microcell fusion, and one of the STFs restored the cellular-senescence program. The concordance of the cellular-senescence-restoring program with the presence or absence of specific DNA fragments of chromosome 2 indicated that the putative cellular-senescence gene was located in 2q32-qter. For more detailed mapping, we constructed mouse A9 cells containing STFs derived from human chromosome 2 tagged with pSTneo at different regions in 2q31-qter. PCR-laddering and FISH analyses were used to identify six clones that contained different STFs. These STFs were transferred into SiHa cells, and one of the three clones that restored cellular senescence contained a small fragment of human chromosome 2. This STF was shown by PCR analysis using 14 human chromosome 2-specific primer pairs to be smaller than 12.2 cM and was mapped to the 2q37 region by FISH analysis with inter-Alu PCR. Beta-galactosidase activity, which is a biomarker of senescent cells, and telomerase activity similar to that found in parental SiHa cells were detected in SiHa microcell hybrids, suggesting that the putative cellular-senescence gene was not involved in a telomerase pathway but rather in an alternate pathway of cellular senescence.

Animals↗
PubMed1998

Repression of the telomerase catalytic subunit by a gene on human chromosome 3 that induces cellular senescence.

The cellular senescence program is controlled by multiple genetic pathways, one of which involves the regulation of telomerase and telomere shortening. The introduction of a normal human chromosome 3 into the human renal cell carcinoma cell line RCC23 caused repression of telomerase activity, progressive shortening of telomeres, and restoration of the cellular senescence program. We attributed the repression of telomerase activity to the marked downregulation of the gene encoding the catalytic subunit of telomerase (hEST2/hTRT) but not another protein component (TP1/TLP1) or the RNA component of telomerase. These results suggest that a senescence-inducing gene on chromosome 3 controls hEST2/hTRT gene expression either directly or indirectly and support the notion that hEST2/hTRT is the major determinant of telomerase enzymatic activity in human cells.

Carcinoma, Renal Cell↗
PubMed1998

Telomerase activity significantly correlates with cell differentiation, proliferation and lymph node metastasis in colorectal carcinomas.

Telomerase activity was examined by the telomeric repeat amplification protocol assay, in a total of 37 colorectal adenocarcinomas, including stages A, B and C according to the Astler and Collier classification, and correlated with clinicopathological features. Of 17 stage C lesions, 13 were positive (76.5%; P<0.01), demonstrating a significant correlation with lymph node metastasis. In contrast, only 6 of 20 stage A and B carcinomas were positive (30.0%), this being significantly lower (P < 0.05). Moderately or poorly differentiated subtypes were more predominant in the telomerase-positive than in the telomerase-negative groups (P< 0.05) with greater elevation of mitotic and Ki-67 labeling indices (P < 0.0001). No significant relation was found between telomerase activity and p53 protein accumulation or Bcl-2 protein expression. The good correlation with tumor staging, lymph node metastasis, differentiation, and mitotic and Ki-67 labeling indices suggests that this parameter might have potential application in estimation of prognosis.

Adenocarcinoma↗
PubMed1998

Functional evidence for involvement of multiple putative tumor suppressor genes on the short arm of chromosome 3 in human oral squamous cell carcinogenesis.

Cytogenetic and restriction fragment length polymorphism (RFLP) analyses have suggested that a putative tumor suppressor genes(s), which may play an important role in the development of human oral squamous cell carcinoma (SCC), is located on the short arm of chromosome 3 (3p). We previously reported that introducing in intact human chromosome 3 into three different oral SCC tumorigenic cell lines completely suppresses the tumorigenicity of each cell line with significant decrease in the in vitro growth rate and morphological changes. To map the tumor suppressor gene(s) on 3p, we have now examined the tumorigenicity of microcell hybrid clones containing various fragments derived from 3p that were introduced by microcell-mediated chromosome transfer. Sixteen hybrid clones were obtained from four successful experiments, and these clones were classified into two groups: 4 fully tumorigenic clones and 12 suppressed phenotype clones. Analyses of the 3p segments in the series of hybrid clones with the use of RFLP or microsatellite markers revealed that the 3p21.2-p21.3 or 3p25 regions or both were consistently retained in the 12 clones with suppressed phenotype but not in the 4 tumorigenic clones. The more proximal 3p13 region also was retained in three nontumorigenic clones. The overall results are fairly compatible with recent evidence that there are three discrete regions on 3p showing frequent allelic losses on oral SCC, and they directly provide functional evidence for the presence of tumor-suppressor genes for oral SCC in these regions. The possibility that three genes, FHIT, VHL, and T beta R-II, recently identified on 3p may be significantly involved in oral SCC development is also discussed.

Animals↗
PubMed1998

Positional cloning of the gene for Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS), also known as ataxia-telangiectasia (AT) variant, is an autosomal recessive disorder characterized by microcephaly, growth retardation, severe combined immunodeficiency and a high incidence of lymphoid cancers. Cells from NBS patients display chromosome instability, hypersensitivity to ionizing radiation and abnormal cell-cycle regulation after irradiation, all of which are characteristics shared with AT. Recently, the NBS locus was mapped at 8q21 by two independent approaches, complementation studies and linkage analysis. Here, we report the positional cloning of the NBS gene, NBS1, from an 800-kb candidate region. The gene comprises 50 kb and encodes a protein of 754 amino acids. The amino-terminal region of the protein shows weak homology to the yeast XRS2, MEK1, CDS1 and SPK1 proteins. The gene is expressed at high levels in the testes, suggesting that it might be involved in meiotic recombination. We detected the same 5-bp deletion in 13 individuals, and conclude that it is likely to be a founder mutation.

Amino Acid Sequence↗
PubMed1998

Epigenetic reprogramming of the human H19 gene in mouse embryonic cells does not erase the primary parental imprint.

BACKGROUND: Genomic imprinting in mammals is thought to result from epigenetic modifications to chromosomes during gametogenesis, which leads to differential allelic expression during development. There is a requirement for an appropriate experimental system to enable the analysis of the mechanisms of genomic imprinting during embryogenesis. RESULTS: To develop a novel in vitro system for studying the molecular basis of genomic imprinting, we constructed mouse cell lines containing either a paternal or maternal human chromosome 11, by microcell-mediated chromosome transfer. Allele-specific expression and DNA methylation studies revealed that the imprinting status of the human H19 gene was maintained in mouse A9 mono-chromosomal hybrids. Each parental human chromosome was introduced independently into mouse near-diploid immortal fibroblasts (m5S) and two embryonal carcinoma (EC) cell lines (OTF9-63 and P19). The paternal allele of human H19 remained in a repressed state in m5S cells, but was de-repressed in both EC cells. The paternal H19 allele was demethylated extensively in OTF9-63 cells, whereas the only alteration in P19 hybrids was de novo methylation on both alleles in the 3' region. Following in vitro differentiation, the expressed paternal H19 allele was selectively repressed in differentiated derivatives of EC hybrids. CONCLUSION: These results indicated that human imprint marks could function effectively in mouse cells, and that the imprinting process was epigenetically reprogrammed in embryonal carcinoma cells, without erasure of the primary imprint that marked the parental origin. Therefore, these mono-chromosomal hybrids could provide a valuable in vitro system to study the mechanisms involved in the regulation of imprinted gene expression.

Animals↗
PubMed1998

Ectopic expression of wnt-5a in human renal cell carcinoma cells suppresses in vitro growth and telomerase activity.

Loss of genes located on chromosome 3p has been reported in many different types of human cancers, including renal cell carcinoma. Previous studies using a nontumorigenic human renal cell carcinoma cell line (RCC23) established from a stage III nonpapillary carcinoma with a loss of heterozygosity on 3p showed that microcell hybrids containing an introduced intact chromosome 3 resulted in a more differentiated phenotype including restored cellular senescence and repression of telomerase activity. Human wnt-5a has been cloned and mapped to chromosome 3p14-21. We have stably transfected human wnt-5a into RCC23 cells which results in in vitro growth suppression and repression of telomerase activity in a manner similar to that found in microcell hybrids containing an introduced intact chromosome 3.

Carcinoma, Renal Cell↗
PubMed1998

Telomerase activity and microsatellite instability in colorectal cancer and adenoma.

In order to examine their roles in carcinogenesis or in progression of colorectal carcinoma, we investigated telomerase activity and microsatellite instability in 67 non-familial colorectal cancers and in 18 adenomas. The incidence of detectable telomerase activity increased from 22% of normal colorectal mucosas adjacent to carcinoma, and 33% of adenomas, to 75% of carcinomas. On the other hand, the incidence of detectable microsatellite instability in carcinomas (30%) was almost the same as in adenomas (22%). No significant correlation was detected in the incidence of telomerase activity and microsatellite instability in carcinomas or in adenomas. Moreover, the incidence of telomerase activity and microsatellite instability did not increase during the progression of carcinomas. These results indicate that telomerase activity and microsatellite instability are independent events in colorectal carcinogenesis, and that telomerase activity and microsatellite instability are not correlated with the progression of colorectal carcinoma. However, in 13 multiple cancers, the incidence of telomerase activity (92%) and the incidence of microsatellite instability (54%) was higher than that of telomerase activity (70%) and that of microsatellite instability (24%) in 54 sporadic cancers. Moreover, the incidence of telomerase activity and that of microsatellite instability in adenomas with carcinomas (45% and 36% respectively) was higher than that of telomerase activity and microsatellite instability in adenomas without carcinomas (14% and 0% respectively). These results indicate that telomerase activity and microsatellite instability may play an important role in multicentric carcinogenesis in colorectal carcinoma.

Adenoma↗
PubMed1998

[Telomerase repressor genes].

Genes involved in the senescence program have been mapped to over ten different genetic loci using microcell fusion to introduced human chromosomes and restore the senescence program. Multiple pathways of cellular senescence have also been demonstrated by chromosome transfer, indicating that the functions of the mapped senescence genes are probably difference. Restoration of cellular senescence by chromosome 3p or 10p is associated with repression of telomerase function in human tumor cell lines, and other chromosomes induced cellular senescence without telomerase suppression. This evidence suggests that telomerase suppression and thereby telomere shortening is one of several pathways involved in cellular senescence.

Animals↗