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Biomedical subjects

T Kanaya

Publications and source records attributed to T Kanaya.

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

Expression of human telomerase subunits in ovarian malignant, borderline and benign tumors.

Telomerase activity is involved in the maintenance of telomere length and is thought to be required for cellular immortality and oncogenesis. Three major subunits composing telomerase, human telomerase RNA (hTR), telomerase-associated protein (TPI) and human telomerase catalytic subunit (hTERT), have been identified. However, their functions and the regulatory mechanisms by which telomerase is activated have not been fully determined. In the present study, a total of 35 epithelial ovarian cancers, 5 ovarian low potential malignancies (LPM), 11 ovarian benign cysts and 12 normal ovaries, as well as various cell lines derived from ovarian cancers, were examined for the expression of hTR, TPI mRNA and hTERT mRNA. Correlations of expression with telomerase activity were evaluated. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that hTR and TPI mRNA were expressed in more than 80% of ovarian cancers, LPM, ovarian cysts and even in normal ovaries. However, hTERT mRNA was observed only in ovarian cancers, most of which exhibited telomerase activity. Normal ovarian tissues, ovarian cysts and LPM, most of which had no telomerase activity, did not express hTERT. Five telomerase-positive ovarian cancer cell lines expressed each of the telomerase subunits, whereas 2 telomerase-negative normal primary fibroblast cell lines expressed TPI mRNA and hTR, but not hTERT mRNA. There was a significant correlation of telomerase activity with hTERT mRNA expression but not with TPI or hTR expression. Expression of hTERT is thus specific to cancer lesions and appears to be a rate-limiting determinant of the enzymatic activity of human telomerase. Up-regulation of hTERT may play a critically important role in the development of ovarian cancers.

Adenocarcinoma, Clear Cell↗
PubMed1999

Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells.

Telomerase activation is thought to be a critical step in cellular immortalization and carcinogenesis. Of the three major subunits comprising human telomerase, human telomerase catalytic subunit (hTERT) has been shown to be a rate-limiting determinant of the enzymatic activity of human telomerase. However, little is known concerning how expression of hTERT is regulated in human cells. To identify the regulatory elements controlling hTERT gene expression, approximately 3.5 kb of the 5'-flanking sequence of hTERT was cloned and characterized. The promoter of hTERT was GC rich and lacked both TATA and CAAT boxes. The CapSite Hunting method identified transcription start site 19 bp upstream of the first nucleotide of the published cDNA sequence. Transient expression assays revealed that transcription of hTERT was significantly activated in cancer cell lines but repressed in normal primary cells. Using the fibroblast lineage at various stages of transformation, we found that transcription occurred in strains that had overcome replicative senescence and expressed telomerase activity. Deletion analysis of hTERT promoter identified the 181-bp core promoter region upstream of the transcription start site. Gel shift analysis revealed two major factors binding to core promoter, an E box (CACGTG) binding factor and Sp1. Overexpression of c-Myc resulted in a significant increase in transcriptional activity of the core promoter. These findings suggest that hTERT expression is strictly regulated at the transcription machinery, and that the proximal core promoter containing an E box and Sp1 sites is required for transactivation of hTERT.

Base Sequence↗
PubMed1999

Human telomerase reverse transcriptase as a critical determinant of telomerase activity in normal and malignant endometrial tissues.

Telomerase activation is thought to be essential for cellular immortality and oncogenesis. It is observed in most malignant tumors but not in most normal somatic tissues. Normal human endometrium is, however, known to express significant telomerase activity in a menstrual phase-dependent manner. The 3 major subunits composing telomerase have been identified. Using normal and malignant endometrial tissues, we studied how these components are involved in telomerase activation. A total of 23 endometrial cancers and 32 normal human endometria in various menstrual phases as well as cell lines derived from endometrial cancer were examined for the expression of each telomerase subunit using RT-PCR analysis. Telomerase activity in each sample was determined by the TRAP assay, and the correlation between subunit expression and telomerase activity was examined. RT-PCR analysis revealed that telomerase RNA (hTR) and telomerase-associated protein (TP1) mRNA were constitutively expressed in both normal and malignant endometrial tissues. Expression of human telomerase reverse transcriptase (hTERT) mRNA was observed in most endometrial cancers, while that in normal endometrium depended on the phases of menstrual cycles. Proliferative phase normal endometria expressed hTERT mRNA, while secretory phase endometria did not. There was a strong association between telomerase activity and hTERT expression but not TP1 or hTR expression in both normal and tumor tissues. Five telomerase-positive endometrial cancer cell lines expressed each of the telomerase subunits including hTERT, while 2 telomerase-negative normal primary fibroblast cells expressed TP1 mRNA and hTR, but not hTERT mRNA. Our findings suggest that hTERT is a rate-limiting determinant of enzymatic activity of human telomerase. Since some normal tissues with high regenerative potential can express hTERT, special attention should be paid to the clinical use of hTERT inhibitors as anti-cancer drugs.

Carrier Proteins↗
PubMed1999

Microscopic basis of free-volume concept as studied by quasielastic neutron scattering and positron annihilation lifetime spectroscopy.

We have reexamined the free-volume concept presented by Cohen and Turnbull on the basis of two microscopic quantities: the excess mean-square displacement (f) and the total free volume V(PA,t), of poly- butadiene evaluated from the quasielastic neutron scattering and the positron annihilation lifetime spectroscopy (PALS) data, respectively. Comparing with the viscosity eta we found two relations, eta=eta(0) exp u( 2)(0)/ (f) and eta=eta(0) exp V(*)(PA,0)/V(PA,t)=eta(0) exp V(*)(PA,0)/v(PA,f), where u(2)(0), V(*)(PA,0) and v(*)(PA,0) are the critical values for the mean-square displacement, the total PALS free volume, and the PALS free volume per molecule, respectively, and further v(*)(PA,0)=V(*)(PA,0)/N, N being the total number of molecules or segments. On the basis of these relations, we discuss the microscopic basis of the free-volume theory. The experimentally evaluated critical values u(2)(0) and v(*)(PA,0) are much larger than the average values of (f) and v(PA,f) calculated from the distributions. This has been explained from the low probability of escaping motions from a molecular cage. The free volume per monomer and the free-volume fraction were calculated from the excess mean-square displacement (f). The former was compared with the free-volume hole obtained by PALS, suggesting that 22 monomers are required for one PALS free-volume hole. The free-volume fraction obtained from the excess mean-square displacement was found to be 6.4% at 250 K, which is in reasonable agreement with that evaluated from the rheological data (9.0%).

Journal Article↗
PubMed1999

DNA replication of human papillomavirus type 31 is modulated by elements of the upstream regulatory region that lie 5' of the minimal origin.

The viral replication factors E1 and E2 of papillomaviruses are necessary and sufficient to replicate plasmids containing the minimal origin of DNA replication in transient assays. Under physiological conditions, the upstream regulatory region (URR) governs expression of the early viral genes. To determine the effect of URR elements on E1 and E2 expression specifically, and on the regulation of DNA replication during the various phases of the viral life cycle, we carried out a systematic replication study with entire genomes of human papillomavirus type 31 (HPV31), a high-risk oncogenic type. We constructed a series of URR deletions, spacer replacements, and point mutations to analyze the role of the keratinocyte enhancer (KE) element, the auxiliary enhancer (AE) domain, and the L1-proximal end of the URR (5'-URR domain) in DNA replication during establishment, maintenance, and vegetative viral DNA amplification. Using transient and stable replication assays, we demonstrate that the KE and AE are necessary for efficient E1 and E2 gene expression and that the KE can also directly modulate viral replication. KE-mediated activation of replication is dependent on the position and orientation of the element. Mutation of either one of the four Ap1 sites, the single Sp1 site, or the binding site for the uncharacterized footprint factor 1 reduced replication efficiency through decreased expression of E1 and E2. Furthermore, the 5'-URR domain and the Oct1 DNA binding site are dispensable for viral replication, since such HPV31 mutants are able to replicate efficiently in a transient assay, maintain a stable copy number over several cell generations, and amplify viral DNA under vegetative conditions. Interestingly, deletion of the 5'-URR domain leads to increased transient and stable replication levels. These findings suggest that elements in the HPV31 URR outside the minimal origin modulate viral replication through both direct and indirect mechanisms.

Binding Sites↗
PubMed1998

hTERT is a critical determinant of telomerase activity in renal-cell carcinoma.

Telomerase is a ribonucleoprotein enzyme which stabilizes chromosomal structure, thereby inducing cellular immortality. Three major subunits composing telomerase complex have been cloned, designated hTR (human telomerase RNA), TPI (telomerase-associated protein I), and hTERT (human telomerase reverse transcriptase). In the present study, a total of 36 renal-cell carcinomas (RCC) and adjacent normal tissues, as well as cell lines derived from RCC or normal kidney, were examined for the expression of each telomerase subunit and telomerase activity. RT-PCR analyses revealed that hTR and TP I mRNA were constitutively expressed both in tumor and in normal tissues. In contrast, hTERT mRNA was expressed in most tumors, but not in normal tissues. Telomeric-repeat-amplification-protocol (TRAP) assay revealed that more than 80% of RCC tumor tissue exhibited telomerase activity, while none of the adjacent normal tissue did. There was a significant association of telomerase activity with expression of hTERT mRNA, but not with TPI mRNA or hTR expression. Two cell lines, derived from RCC and cervical cancer, expressed telomerase activity and hTERT mRNA, while normal renal cortical epithelial cells expressed neither of them. These findings suggest that hTERT plays a critical role in determining the enzymatic activity of human telomerase, and that up-regulation of hTERT probably plays a role in the progression of human cancers.

Adult↗
PubMed1998

Expression of human telomerase subunits and correlation with telomerase activity in cervical cancer.

Activation of telomerase and stabilization of telomeres are thought to be required for both cellular immortality and oncogenesis. Three major components of human telomerase, human telomerase RNA (hTR), telomerase-associated protein (TP1/TLP1), and human telomerase catalytic subunit (hTRT/hEST2), have been identified recently. However, it remains unclear what roles these subunits play in the regulation of telomerase activity. In the present study, a total of 25 cervical cancers and 14 normal cervices as well as various cell lines derived from cervical cancer were examined for the expression of hTR, TP1 mRNA, and hTRT mRNA, and the correlations between expression of these and telomerase activity were evaluated in 23 cancers and 14 normal cervices. Reverse transcription-PCR analysis revealed that hTR and TP1 mRNA were commonly expressed in cancers and noncancerous tissues. However, hTRT mRNA was observed only in cervical cancers and cell lines, and more than 80% of cervical cancers expressed it, whereas neither normal cervical tissues nor normal primary fibroblast cells did. There was a strong correlation of telomerase activity with hTRT mRNA expression but not with TP1 or hTR expression. Cervical exfoliated cells were subjected to reverse transcription-PCR analysis for detection of hTRT mRNA, and approximately 70% of cervical cancers were positive for such expression. These findings provide strong evidence that expression of hTRT is a rate-limiting determinant of the enzymatic activity of human telomerase and that up-regulation of hTRT expression may play a critical role in human carcinogenesis. Our findings also indicate that detection of hTRT mRNA is useful for cytological screening for cervical cancer.

Carrier Proteins↗
PubMed1998

Telomerase activity in cervical cancer is quantitatively distinct from that in its precursor lesions.

Studies using the telomeric repeat amplification protocol (TRAP) assay have demonstrated telomerase activity not only in cancers but also in non-cancerous lesions. However, quantitative differences in activity between both lesions have not been examined. In the present study, using a stretch PCR assay, telomerase activity was analyzed quantitatively in cervical cancer, its precursor squamous intra-epithelial lesions (SILs) and normal cervix. In stretch PCR assay, telomerase activity was expressed in relative units vs. control activity from C33A cells (100 units). Mean telomerase activities in cervical cancer, SIL and normal cervix were 72+/-35 units, 18+/-17 units and 7+/-4 units, respectively, suggesting that telomerase activity in cancer lesions was quantitatively distinct from that in pre-malignant lesions, which may mean a much more pronounced activation of telomerase in cancers than in SIL. Our findings also suggest that stretch PCR assay can distinguish telomerase activity in cancer from that in non-cancerous lesions and may be useful for the differential diagnosis of cancer lesions.

Adenocarcinoma↗
PubMed1998

Ovarian endometrioid adenocarcinoma with ectopic production of alpha-fetoprotein.

alpha-Fetoprotein (AFP) is well known as a tumor marker of ovarian endodermal sinus tumor or embryonal carcinoma in gynecological malignancies. However, AFP production is extremely rare in ovarian epithelial cancers. Here we report a case of a 53-year-old woman with an AFP-producing ovarian endometrioid adenocarcinoma. The serum AFP level was elevated up to 2759 ng/ml preoperatively, with a subsequent decrease to the normal range after treatment. Histological examination of the tumor revealed a well-differentiated endometrioid adenocarcinoma with small foci of clear cell components. None of endodermal sinus tumor, hepatoid carcinoma, or embryonal carcinoma components were observed. Immunohistochemical analysis revealed that AFP was expressed in the cytoplasm of the endometrioid glandular lesions, but not in the clear cell components. This is probably the first case of a pure type of ovarian endometrioid adenocarcinoma with significant levels of AFP expression.

Adenocarcinoma↗
PubMed1998

Expression of telomerase activity in human endometrium is localized to epithelial glandular cells and regulated in a menstrual phase-dependent manner correlated with cell proliferation.

Telomerase activity is observed in most malignant tumors and germ cells, whereas normal somatic cells usually do not express it. Human endometrium is composed of glandular and stromal components and exhibits dramatic changes in proliferative activity during the menstrual cycle, which is exquisitely regulated by estrogen function. We previously reported that normal human endometrium expresses telomerase activity. However, it remains unclear which of the above components are the major sources of telomerase activity and how levels of telomerase activity are regulated over the menstrual cycle. Quantitative analysis of telomerase activity revealed that it changes dramatically over the course of the menstrual cycle and is strictly regulated in a menstrual-phase-dependent manner. Maximal activity equivalent to that in endometrial cancer was present in late proliferative phase, and minimal activity in late secretory phase. Postmenopausal endometrium and endometrium treated with anti-estrogen drugs exhibited decreased telomerase activity. Testing isolated epithelial glandular cells and stromal cells, we found that telomerase activity was localized to epithelial glandular cells. In situ RNA hybridization analysis also revealed epithelial-specific expression of human telomerase RNA. In vitro analysis of cultured epithelial cells demonstrated that telomerase activity is correlated with epithelial proliferation but not affected by estrogen treatment. These findings suggest that expression of telomerase activity is specific to epithelial cells and linked to cell proliferative status. The involvement of estrogen in telomerase regulation remains to be elucidated.

Cell Division↗
PubMed1998

Ruptured aneurysm of the ductus diverticulum into the pulmonary artery in a man: a successful repair.

Aneurysm of the ductus diverticulum in the adult is rare and its rupture is fatal. A 75-year-old man presented with congestive heart failure that suddenly occurred with a continuous murmur. Angiography showed a left-to-right shunt through a large thrombosed aneurysm of the ductus diverticulum (6 cm), and the pulmonary-to-systemic flow ratio was 2.6. Patch closure of the orifice (3x4 cm) of the aneurysm and repair of the perforated pulmonary artery were done emergently under hypothermic cardiopulmonary bypass with selective cerebral perfusion. He recovered uneventfully. Early recognition and early intervention should be indicated in this otherwise fatal condition.

Aged↗
PubMed1998

Expression of human telomerase subunits and correlation with telomerase activity in urothelial cancer.

The activation of telomerase and stabilization of telomeres are thought to be required for cellular immortality and oncogenesis. Three major components of human telomerase--human telomerase RNA (hTERC), telomerase-associated protein (TEP1), and human telomerase catalytic subunit (hTERT)-have recently been identified. However, the roles played by these subunits in the regulation of telomerase activity are still unclear. In the present study, a total of 37 urothelial cancers, including one metastatic lesion, and adjacent normal tissues as well as cell lines derived from bladder cancers were examined for the expression of each telomerase subunit. Reverse transcription-PCR analysis revealed that more than 90% of urothelial cancers expressed hTERT mRNA, whereas less than 20% of normal adjacent tissues did. In contrast, hTERC and TEP1 mRNA were commonly expressed in both cancers and normal tissues. All of the three cell lines derived from bladder cancer expressed each of the telomerase subunits, whereas the two normal primary fibroblast cell lines expressed hTERC and TEP1 mRNA but not hTERT mRNA. Telomerase activity was examined using telomeric repeat amplification protocol assay. All of the cancers examined exhibited telomerase activity, whereas only 2 of 12 normal tissues exhibited weak activity. There was a significant association of telomerase activity with hTERT mRNA expression but not with hTERC or TEP1 mRNA expression. These findings provide strong evidence that the expression of hTERT is a rate-limiting determinant of the enzymatic activity of human telomerase and that the up-regulation of hTERT expression may play a critical role in human carcinogenesis.

Aged↗
PubMed1998

Detection of human telomerase reverse transcriptase messenger RNA in voided urine samples as a useful diagnostic tool for bladder cancer.

Activation of telomerase and stabilization of telomeres are thought to be required for cellular immortality and oncogenesis. Telomerase activity is detected in >90% of various cancers, including urothelial cancers. Of the three subunits comprising telomerase complex, human telomerase reverse transcriptase (hTERT) is a rate-limiting determinant of the enzymatic activity of telomerase. In the present study, spontaneously voided urine specimens from 33 patients with bladder cancer and 26 without bladder lesions were examined for the expression of hTERT mRNA, and the usefulness of detecting hTERT mRNA in urine samples for screening of bladder cancer was evaluated. RT-PCR analysis revealed that approximately 80% of urinary sediments from patients with bladder cancer expressed hTERT mRNA, regardless of clinical stage or pathological grade, whereas only 4% of sediments from patients without urothelial lesions did. Interestingly, hTERT mRNA expression was observed, even in some urine samples from bladder cancer patients with negative urinary cytology. These findings suggest that the expression of hTERT in urine sample may be a useful diagnostic marker for bladder cancer.

Aged↗
PubMed1997

Expression of telomerase activity in human chorion.

Telomerase activation is required for cellular immortalization and is found in most malignant tumors. Normal somatic cells are generally telomerase-negative, except for stem cells in renewing tissues. During pregnancy, human trophoblast continues to proliferate and acts as proliferating stem cells for the development of chorion and the formation of placenta. In the present study, a total of 105 chorions from placentas at various weeks of gestation were examined for telomerase activity using the telomeric repeat amplification protocol (TRAP) assay. Twenty-five of 33 (76%) normal early chorions at 5 to 9 weeks gestation were telomerase-positive. Chorions from early spontaneous abortions also exhibited telomerase activity but at a low level. In contrast, only 2 (4%) late chorions at 34 to 41 weeks gestation expressed telomerase activity. Significant telomerase activity was observed in trophoblast cell fractions of chorion, demonstrating trophoblast to be the source of the activity. Expression of human telomerase catalytic subunit (hTRT) was observed in early chorions, but not in late placenta, and there was a close correlation between telomerase activity and hTRT expression. In contrast, expression of human telomerase RNA component (hTR) was observed in both early and late chorions and was not liked to telomerase activity. These findings suggest that telomerase activity in chorion is critically regulated over the course of gestation, associated with hTRT expression. The findings of the present study also appear to support the emerging concept that normal somatic cells with stem cell-like characteristics can express telomerase activity.

Choriocarcinoma↗
PubMed1997

The 5' region of the human papillomavirus type 31 upstream regulatory region acts as an enhancer which augments viral early expression through the action of YY1.

Cis-elements which control human papillomavirus early gene expression have previously been localized to sequences in the upstream regulatory region (URR) which are proximal to the E6 open reading frame. These elements include an enhancer element which functions preferentially in keratinocytes as well as promoter elements. The function of the remaining approximate 500-bp region of the URR in regulating viral expression in the high risk papillomaviruses has been largely uncharacterized. In HPV 6, a negative regulator of early expression, or silencer, has been identified in this 5' region of the URR. In this study, we have investigated the role of the 5' portion of the HPV 31 URR in regulating viral expression. Sequences in this region were found to exert a minimal negative effect on homologous or heterologous promoters. In contrast, a 128-bp sequence within this region was found to exhibit enhancer activity on heterologous and homologous promoters. This enhancer also augmented the activity of the previously identified minimum keratinocyte enhancer. The cellular factors, YY1 and TEF-1, were determined by DNase I footprint analysis and electrophoretic mobility shift assays (EMSAs) to bind the 128-bp enhancer. The binding of YY1 to two of these sites was found to be important for enhancer activity.

Base Sequence↗
PubMed1997

Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene.

Infection by a baculovirus (Bombyx mori nuclear polyhedrosis virus, BmNPV) in silkworm (Bombyx mori) larvae is highly efficient as an expression system for the production of useful proteins. However, the amount of the protein of interest expressed tends to decrease in the later stages of infection presumably due, in part, to a proteinase produced in the larval haemolymph. The N-terminal amino acid sequence of a proteinase purified from the haemolymph of BmNPV-infected larvae was identical to the internal amino acid sequence of the viral cysteine proteinase gene of BmNPV, suggesting that the cysteine proteinase in the haemolymph originated from the BmNPV gene. We constructed a mutant virus (CPd) which had a deletion in the cysteine proteinase gene. No proteinase activity corresponding to this proteinase was detected in the haemolymph of silkworm larvae infected with CPd. The firefly luciferase and the human growth hormone genes were separately introduced into CPd under control of the polyhedrin promoter. These constructs produced these proteins very efficiently, because of a greatly reduced degree of degradation of these proteins. A BmNPV vector system using CPd enhances the stability of foreign expressed proteins, especially for those that are cysteine proteinase-sensitive.

Animals↗
PubMed1997

Expression of AP1 during cellular differentiation determines human papillomavirus E6/E7 expression in stratified epithelial cells.

E6 and E7 oncoproteins of human papillomavirus (HPV) play significant roles in the pathogenesis of cervical cancer. However, the pattern of E6/E7 expression during the productive virus life cycle in differentiating epithelia of the uterine cervix remains unclear. In addition, little is known about the cellular factors regulating E6/E7 expression in differentiating epithelia. In the present study, using transient expression assays and DNA binding assays, we demonstrated that E6/E7 transcription is critically regulated by the cellular factor AP1, a Jun/Fos heterodimer complex. Immunohistochemical analyses of various uterine cervical lesions showed AP1 expression in lower cell layers of normal cervix and low-grade cervical intraepithelial neoplasia (CIN), while it was detected throughout all layers in high-grade CIN and invasive cancer. In situ RNA-RNA hybridization analyses of organotypic raft culture specimens of an HPV-31-containing cell line revealed that E6/E7 transcripts were expressed in most cell layers, with reduced expression in differentiated cells. This pattern of HPV expression correlated with the pattern of AP1 expression detected by immunohistochemical analyses. These findings suggest that E6/E7 expression in differentiating epithelia is dependent on AP1, which appears to be associated with proliferative activity of the cells. Since E6/E7 expression induces cell proliferation, co-expression of AP1 and E6/E7 in undifferentiated cell layers might create a positive regulatory loop, probably contributing to maintenance of initial HPV infection and subsequent activation in basal and suprabasal cell layers.

Binding Sites↗