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Biomedical subjects

N Wake

Publications and source records attributed to N Wake.

At least 37 records · Page 2Linked to original sources
PubMed1999

WAF1 genotype and endometrial cancer susceptibility.

The WAF1 protein, which is a downstream mediator of p53, functions as a universal inhibitor of cyclin-dependent kinases. The functional link between p53 and WAF1 suggests the possibility that alteration in WAF1 function constitutes an alternative mechanism to p53 inactivation. However, there are few reports describing somatic mutations of the WAF1 gene in various human malignancies. A polymorphism in the WAF1 gene, a C-to-A transversion at codon 31 resulting in the change of a serine (Ser) to an arginine (Arg), is well known. We found this substitution in 42 of 54 endometrial carcinoma patients. Allele frequency was 0.44/0.56 for the codon 31 polymorphism (Ser/Arg), the difference of allele frequency between patients and normal controls being significant (0.59/0.41 in normal controls). In addition, individuals carrying the codon 31 Arg allele had a tendency to develop histologically high-grade (odds ratio, 6. 11) and clinically advanced tumors. We investigated the association of the Arg allele with the known risk factors of endometrial carcinomas. Statistical analyses of 42 cases and 32 controls carrying the codon 31 Arg allele identified hypertension (odds ratio, 4.33) and family history of cancer (odds ratio, 2.81) as positive risk factors. This implies that these two parameters may be associated with a tendency to develop endometrial carcinomas in individuals carrying the codon 31 Arg allele of the WAF1 gene.

Alleles↗
PubMed1999

Gestational choriocarcinoma whose responsible pregnancy was a complete hydatidiform mole identified by PCR analysis with new sequence tagged site primers.

We report a case where the pregnancy responsible for a gestational choriocarcinoma was not the antecedent pregnancy or the second normal term delivery, but a complete hydatidiform mole that had advanced to clinically invasive mole. This responsible pregnancy was identified by polymerase chain reaction analysis (PCR). PCR analysis was performed by using five new sets of sequence-tagged site (STS) primers on four chromosomes (chr. 1, D1S225; chr. 3, D3S1744; chr. 12, D12S1090; chr. 18, D18S849 and D18S877). The constitution of alleles of choriocarcinoma was shown to be almost identical with that of the husband on every marker. The allele patterns of choriocarcinoma on D3S1744 and D12S1090 were not observed with DNA from the patient. The band pattern originating from molar DNA was also identical with those of the husband and choriocarcinomas on D18S849 and D1S225.

Choriocarcinoma↗
PubMed1999

Photodynamic antisense regulation using psoralen-conjugated oligo(nucleoside phosphorothioate)s (I). Growth regulation of cervical carcinoma cells.

To increase the antisense regulatory effect of oligo(nucleoside phosphorothioate)s (S-Oligo), a photo-crosslinking reagent, 4, 5', 8-trimethylpsoralen, was used in this study. Psoralen-conjugated oligo(nucleoside phosphorothioate) (Ps-S-Oligo) complementary to the human papillomavirus type 18 (HPV18) mRNA drastically inhibited the cellular proliferation of cervical cancer cells only upon UVA-irradiation. In contrast, Ps-S-Oligos with mismatched sequences and scrambled one showed lesser inhibitory effects than that with matched one. These results suggest that psoralen-conjugated antisense S-Oligo has significant potential to regulate gene expression upon UVA-irradiation.

Cell Division↗
PubMed1999

Mutation analysis of the Smad3 gene in human ovarian cancers.

The Smad3 gene is a member of the Smad family, vertebrate homologues of Drosophila Mad, and its gene product is a cytoplasmic element in the transforming growth factor-beta (TGF-beta) signaling pathway. Mutations in TGF-beta receptors and their cytoplasmic elements of transduction signals commonly accompany various cancers. Using PCR-SSCP analysis we searched for the presence of Smad3 gene mutations in 36 human ovarian cancers, and found that 15 cases (41. 7%) had a polymorphism at codon 103. Because this mutation was not accompanied by amino acid replacement, the present results show that the mutations in the Smad3 gene are unlikely to be involved in human ovarian cancers.

Adolescent↗
PubMed1998

CyclinG contributes to G2/M arrest of cells in response to DNA damage.

To investigate regulation mechanisms of G2/M phase transition, we studied the association of cell cycle progression with p53-dependent p21/waf-1 and cyclinG expression. We used doxorubicin (DOX) and sodium butyrate (NaB) to accumulate p53 protein. DOX treatment resulted in an apparent increase of cells in the G2/M fraction, whereas NaB arrested cells at G1. P53 protein induction in response to DOX accompanied up-regulation of p21/waf-1 and cyclinG expression. However, cyclinG was undetectable in NaB-treated cells. These results implied a putative association between increases in the proportion of cells accumulating in the G2/M fraction and enhanced cyclinG expression. Antisense oligo DNAs (AS) complementary to cyclinG mRNA inhibited the cyclinG protein expression induced by DOX treatment. This inhibition resulted in a marked reduction in the number of cells arrested at G2/M and accumulating at G1. A role for cyclinG in G2/M phase transition control is implied.

Animals↗
PubMed1998

Microsatellite instability and somatic mutations in endometrial carcinomas.

Recently, microsatellite instability (MI) has been demonstrated in some types of human cancers. In this study, we attempted to determine the frequency of MI in endometrial cancers and evaluate whether replication error (RER)-positive phenotype is correlated with known genetic mutations or the aberrations of other pathways in endometrial cancers. Seventy-two primary endometrial cancers were examined for microsatellite instability. Eleven tumors (15%) had RERs at two or more microsatellite loci, suggesting that generalized MI may be a molecular manifestation of endometrial cancers. We next examined whether the MI was associated with changes in the K-ras protooncogene, p53 tumor suppressor gene, and 18q LOH, which were frequently detected in endometrial cancers. The MI did not confer the potential to produce point mutations in the K-ras gene or 18q LOH, whereas the data were insufficient to identify the correlation between MI and p53 mutations in the cancers. These results suggest the presence of multiple mutation subsets that act in a complementary fashion in endometrial cancer development.

Chromosomes, Human, Pair 18↗
PubMed1998

Oncogenic Ras modulates epidermal growth factor responsiveness in endometrial carcinomas.

Since the majority of endometrial carcinomas do not contain any detectable ras mutations, the precise contribution of aberrant Ras function, if any, to endometrial carcinoma development remains to be determined. Since there is considerable evidence that Ras transformation is associated with a decreased requirement for growth factors, we compared the growth response of endometrial carcinoma cells harbouring wild-type (Ishikawa cells) or mutated (HHUA cells) K-ras to epidermal growth factor (EGF). K-ras mutation did not significantly affect the level of the EGF receptor (EGFR) expressed in these carcinoma cells. EGF could stimulate the growth of Ishikawa, but not HHUA cells. Furthermore, EGF caused elevation of Ras-GTP levels in Ishikawa, but not HHUA cells. However, the introduction of mutated, but not normal, K-ras into Ishikawa cells rendered them non-responsive to EGF growth stimulation. Thus, the presence of mutated K-ras alone modulated the growth response of endometrial carcinoma cells to EGF. An inhibitor of the EGFR tyrosine kinase activity could prevent soft agar colony formation of Ishikawa cells, but not HHUA or mutant K-ras(12V)-transfected Ishikawa cells. Taken together, these results suggest that mutated K-ras causes a loss of responsiveness to EGF stimulation and that EGFR function is dispensable for the growth of mutant Ras-positive endometrial carcinoma cells.

Apoptosis↗
PubMed1998

Requirement of estrogen receptor expression and function for [12Val] K-Ras-mediated NIH3T3 cell transformation.

We investigated the biological significance of estrogen receptors (ERs) in NIH3T3 cell transformation by the [12Val] K-Ras mutant. This mutant enhanced the steady-level and transcriptional activity of ER. Coexpression of the progesterone receptor with mutant K-Ras led to suppression of tumorigenicity and inhibition of the activation of ER. The antisense oligomers complementary to the ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation.

Cell Transformation, Neoplastic↗
PubMed1998

Involvement of IGF2 and H19 imprinting in choriocarcinoma development.

Complete hydatidiform mole is an abnormal pregnancy characterized by grossly swollen villi in the absence of a fetus (Kajii and Ohama 1997, Wake et al., 1978, Jacobs et al., 1980). It is well known that this abnormal pregnancy product is androgenetic in origin. The entire genome of the molar conceptus is paternally derived. The majority of moles result from fertilization of an empty egg by haploid sperm. The paternally derived haploid set then duplicated without cytokinesis and restores diploidy. Invariably, this class of moles has a 46, XX karyotype and is completely homozygous for genetic markers. Fertilization of an empty egg by two sperms is responsible for the remaining case. These moles show a mixture of homozygous and heterozygous patterns of paternally derived genetic markers. Although complete mole is usually a benign process, 10 to 20 percent of cases leads to either invasive mole or choriocarcinoma. This propensity to malignancy has to associate with the genetic features shown in the mole, that imply the formation of homozygosity and the selective inheritance of paternal genome. It has been described in various human malignancies that both genetic features associate with the inactivation of tumor suppressor genes. Homozygosity would lead to the inactivation of tumor suppressor gene in the mole by a signal event occurred on sperm DNAs. In turn, paternal transmission would result in the silencing of particular tumor suppressor genes. Thus, tumor suppressor genes inactivated either by homozygosity formation or by paternal transmission would be involved in the pathogenesis of choriocarcinoma. Both the maternal and paternal genomes are necessary for normal embryonic development in mammals. Parental-origin-specific functional differences between two alleles, known as genomic imprinting, seem to be exploited for a regulatory mechanism crucial for the proper development of both embryonic and extraembryonic tissues. The list of imprinted genes identified is growing rapidly lately in mouse and man (DeChiara TM et al., 1991, Barlow et al., 1991, Bartolomei MS et al., 1991, Leff SE et al., 1992, Hatada I et al., 1993, Giddings SJ et al., 1994, Hayashizaki Y et al., 1994, Villar AJ et al., 1994, Guillemot F et al., 1995). Among these, IGF2 and H19 tightly linked on human chromosome 11 are of special interest because of their reciprocal imprinting and possible association with certain malignancy and congenital abnormalities. The IGF2 gene expressed from the paternally derived allele (Ohlsson et al., 1993, Giannoukalis et al., 1993), whereas the H19 gene is expressed from the maternally derived allele (Rachmilewitz et al., 1992, Zhang et al 1993, Ferguson-Smith et al., 1993).

Alleles↗
PubMed1997

Contribution of enhanced transcriptional activation by ER to [12Val] K-Ras mediated NIH3T3 cell transformation.

We investigated the biological significance of estrogen receptors (ER) in NIH3T3 cell transformation by the [12Val] K-Ras mutant. This mutant enhanced the steady state level of ER. Cells expressing mutant K-Ras (K12V cell) were tumorigenic. To determine the role of ER accumulation in Ras-transformed cells, we developed cells (KwtER cells) that overexpressed both wild-type (wt) K-Ras and ER, and found these cells were also tumorigenic. E2 stimulated the transcriptional activity by ER dominantly in K12V cells. However, only partial activation of ER by E2 was seen in KwtER cells. In the presence of 10% serum in media, the activation of ER appeared only in transformed KtwER and K12V cells, suggesting that two independently transmitted signals, the E2-ER binding and the ER-AF1 activation, are necessary for ER activation and that the dominant activation of ER might be involved in Ras-mediated cell transformation. Co-expression of progesterone receptor (PR) with mutant K-Ras led to suppression of tumorigenicity and inhibition of the activation of ER. The antisense oligomers complementary to the ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation.

3T3 Cells↗
PubMed1997

Human chromosome 7 carries a putative tumor suppressor gene(s) involved in choriocarcinoma.

Choriocarcinoma developed from a complete hydatidiform mole has an unique genetic feature that involves monoallelic contribution from the paternal genome. To determine the chromosome carrying putative tumor suppressor gene(s), microcell-hybrids were isolated following fusion of choriocarcinoma cells with microcells from mouse A9 cells containing a single human chromosome (1, 2, 6, 7, 9 or 11). Microcell-hybrids with the introduction of chromosome 7 were suppressed or modulated for tumorigenicity and exhibited altered in vitro growth properties. Introduction of chromosomes 1, 2, 6, 9 or 11 had no effect. Tumorigenic revertants isolated from microcell-hybrids with the introduced chromosome 7 contains reduced numbers of chromosome 7. These findings suggest that chromosome 7 contains a putative tumor suppressor gene(s) for choriocarcinoma. Alterations in tumorigenic phenotypes seen in microcell-hybrids were not associated with the presence of either ERV3 or H-plk locus located on the introduced chromosome 7, indicating the putative tumor suppressor gene(s) is outside of ERV3 and H-plk gene loci. Furthermore, we obtained evidence to define a critical region on chromosome 7 (7p12-7q11.23) that was frequently lost in surgically removed choriocarcinoma tissues and cell lines. Using a panel of microsatellite markers, biallelic deletions were observed, which strongly suggests the presence of a tumor suppressor gene(s) within this critical region.

Animals↗
PubMed1997

Alterations of the p16INK4A gene in human ovarian cancers.

The p16INK4A gene, which encodes the cell-cycle regulatory protein cyclin-dependent kinase 4 inhibitor, is a putative tumor-suppressor gene. We examined p16 gene alterations in 30 primary ovarian cancers and 11 ovarian cancer cell lines. Five of the primary cancers (16.7%) had lost both p16INK4A genes. In addition, four cancers (13.3%) contained five kinds of missense mutations and a one-base deletion. Three cell lines had homozygous deletions of p16 genes, and one cell line had multiple intragenic mutations. There was also suppressed transcription of the p16 gene in one cell line. Some point mutations occurred in the conserved ankylin consensus region. These observations suggest that p16 is a functional target for ovarian carcinogenesis and that p16 alterations occurred in the primary cancers.

Adenocarcinoma↗
PubMed1997

Association of IGF2 and H19 imprinting with choriocarcinoma development.

We studied IGF2 and H19 expression, and methylation status of H19 gene in androgenetic moles and choriocarcinomas. The human placentae were examined similarly as a control. The CpG sites analyzed for methylation covered the 5' portion and the entire coding regions of H19. Although the paternal IGF2 and the maternal H19 allele were exclusively transcribed in full-term placentae, both H19 alleles were active in early placentate of 6-8 weeks gestation. The level of H19 expression in the mole was similar to that in normal placentae, which is compatible with the finding that half of the H19 gene was methylated and the remaining one was hypomethylated en masse in the complete mole. These imply the importance of regulating the level of H19 transcription not only for normal embryogenesis but also for the development of androgenetic moles. Choriocarcinomas were characterized by a low expression of IGF2 and a high expression of H19 with the transcripts being apparently intact in size. Biallelic expression of IGF2 or H19 was found frequently but not consistently in choriocarcinomas. Contrary to expectation, enhanced H19 expression was accompanied by hypermethylation of CpG sites over the entire gene region, apparently being at variance with the finding in normal placentae and androgenetic moles. The hypermethylation of CpG sites was also recognized in choriocarcinoma specimens surgically removed. The active H19 allele was unmethylated in placentae and probably so in androgenetic moles, but it was heavily methylated in choriocarcinomas. These findings provide the possibility that the mutated promoter is responsible for overcoming transcriptional suppression by CpG methylation in the H19 gene.

Blotting, Southern↗
PubMed1996

Poor correlation with loss of heterozygosity on chromosome 17p and p53 mutations in ovarian cancers.

To define the target of chromosome 17p deletions, allelic losses in the 17p11.2 to 13.3 regions of 32 ovarian cancers were investigated. Twenty-one (68%) of 31 informative cancers had deletions on chromosome 17p. None of these 21 cancers involved deletions in the entire chromosome 17p even if deletions of a small chromosome region were infrequent. Of these 21, 17 cancers contained deletions at 17p13.1 or neighboring regions. The remaining 4 cancers with 17p deletions were uninformative for deletions at 17p13.1. Thus, most 17p deletions seemed to target the 17p13.1 region in ovarian cancers. Of the 30 ovarian cancers screened, 6 contained p53 mutations. One p53 allele was lost as a consequence of deletion and the other was mutated in 4 cancers. Seventeen cancers with deletions on 17p showed no evidence of p53 mutations. Thus, deletions on 17p that are common in ovarian cancers are not always accompanied by p53 gene mutations.

Alleles↗
PubMed1995

Suppression of endometrial carcinoma cell tumorigenicity by human chromosome 18.

Presumptive tumor suppressor genes may be localized to specific chromosomes by the procedure of microcell fusion, whereby individual chromosomes derived from normal human cells are introduced into tumor cells. Allelic loss on chromosome 18 is commonly seen in endometrial carcinoma, and the DCC gene on chromosome 18q is a potential human tumor suppressor gene. In this study, we investigated the hypothesis that a gene on chromosome 18, possibly DCC, is capable of suppressing the tumorigenicity of endometrial carcinoma cells. Microcells from the mouse A9 cell clone containing one human chromosome 18 tagged with the pSV2-neo plasmid were fused with the highly tumorigenic endometrial carcinoma cell lines HHUA and Ishikawa, and G418-resistant microcell hybrids containing and extra copy of chromosome 18 were isolated. Clones isolated from the HHUA cell line were completely suppressed for tumorigenicity in nude mice, and clones from the Ishikawa line were suppressed or inhibited for tumorigenicity. In contrast, growth rates in vitro were not significantly affected in clones from either parental cell line. DCC expression was elevated in most of the suppressed hybrids. These results indicate that a gene on human chromosome 18 is capable of suppressing the tumorigenicity of endometrial carcinoma cells, and that DCC is a candidate for this endometrial carcinoma tumor suppressor gene.

Animals↗
PubMed1995

Transcriptional repression of smooth-muscle alpha-actin gene associated with human papillomavirus type 16 E7 expression.

To explore the role of the E7 viral oncogene from human papillomavirus type 16 (HPV 16) in the regulation of cytoskeletal organization, we investigated alterations in particular cytoskeletal components in rat embryonal fibroblasts and three transformants of rat embryonal fibroblast cells produced by transfections with HPV16 E7 alone (TF1), HPV16 E7 plus adenovirus type 5 E1B (TF3), and HPV16 E7 plus activated Ha-ras (TF4). Marked reductions in smooth-muscle (SM) alpha-actin content and disrupted organization of stress fibers detected by anti-SM alpha-actin antibody were evident in all the transformants. These cytoskeletal manifestations were associated with a significant reduction in the mRNAs in these cells. Transcriptional repression by the E7 gene was observed after transient transfection of a chloramphenicol acetyltransferase reporter gene with SM alpha-actin gene promoter. Nucleotides -123 to -39 of the SM alpha-actin gene promoter were required for the HPV16 E7 transcriptional repression as shown by the chloramphenicol acetyltransferase assay. The downregulation of this actin isoform mediated by the E7 oncoprotein may play an important role in cell transformation by HPV16.

3T3 Cells↗
PubMed1995

Malignant trophoblastic neoplasms with different modes of origin.

The genetic origin of 24 trophoblastic neoplasms was determined using PCR polymorphisms. Based on pregnancy history, these tumors included nine postmolar trophoblastic tumors, 12 tumors preceded by live birth or abortion, and three nongestational tumors. Androgenetic origin was defined in eight post-molar trophoblastic tumors, and the remaining one might have arisen from a normal fertilization. Six tumors retained genetic features carried by the homozygous complete mole. Two tumors showed PCR polymorphism compatible with that of the heterozygous complete mole. All 12 tumors in the second class had alleles of both paternal and maternal contribution. However, discordance of sex between the antecedent pregnancy product and the tumor was recognized in three choriocarcinomas. The absence of paternal contribution suggested a parthenogenetic origin of three nongestational choriocarcinomas. The findings that PCR polymorphisms were either homozygous in certain loci or heterozygous in others may mean that the tumor was derived from a germ cell after meiosis I. As a result, at least three subtypes with different modes of origin were demonstrated in the 24 trophoblastic tumors. These findings underscore the importance of precise genetic marker analyses in a large series to clearly identify clinical and biologic characteristics of each subset of tumors.

Base Sequence↗