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J W Shay

Publications and source records attributed to J W Shay.

At least 55 records · Page 3Linked to original sources
PubMed1999

The RNA component of telomerase as a marker of biologic potential and clinical outcome in childhood neuroblastic tumors.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme associated with cellular immortality that may be useful in determining the biologic potential of a tumor. Neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN) are neuroblastic tumors (NTs) that exhibit a spectrum of histologic features and are often associated with unpredictable behavior and clinical outcome. METHODS: The authors investigated the expression of the RNA component of human telomerase (hTR) by in situ hybridization in 32 cases of NTs (including 24 NBs, 4 GNBs, and 4 GNs), using [35S]-UTP labeled single stranded sense and antisense RNA probes. Eight NBs were early stage, 12 NBs were advanced stage, and 4 NBs were Stage IVS, a widely metastatic variant associated with an excellent clinical prognosis. Four NBs had N-myc amplification. In addition, the authors compared a proliferation marker, MIB-1, with hTR expression in a subset of tumors. RESULTS: Thirty of 32 NTs expressed hTR, with expression varying from weak (1+) to intense (4+). Most advanced stage NBs (9 of 12) and only 2 of 8 early stage NBs had moderate to intense (2 to 4-) expression of hTR. The remaining early stage tumors (6 of 8) and 3 of 12 advanced stage NBs had absent or weak expression of hTR (0 to 1+). There was no disease progression in any of the patients with absent or weak expression of hTR. In contrast, 8 tumors (from 7 patients) with moderate to intense expression of hTR in the tumor sections had adverse clinical outcomes, including recurrence, persistent disease, or death. hTR expression in all the Stage IVS tumors was weak, despite the fact that the patients had widely metastatic disease at presentation. The mean hTR score of 3.1 for NBs associated with an adverse outcome (n = 8) was significantly different from the mean hTR score of 1.3 for NBs associated with a favorable outcome (n = 16), P < 0.001. hTR expression in the GNB/GNs was limited to the ganglion cells only; Schwann cells were negative for hTR expression. Stage IVS tumors, which are associated with an excellent outcome, had high MIB-1 but weak hTR expression, indicating that the latter may be a better discriminator of true biologic potential and that hTR levels do not always correlate with cell proliferation. CONCLUSIONS: Increased hTR expression may reflect the potential for aggressive behavior within the spectrum of NTs; conversely, down-regulation of hTR may be useful in identifying subsets with limited capacity for progression and a favorable prognosis.

Adolescent↗
PubMed1999

Telomerase activity does not always imply telomere maintenance.

The forced expression of the catalytic subunit of human telomerase, hTERT, produces telomerase activity, allows telomere maintenance, and extends the cellular life span of IMR90 human lung fibroblasts. The mutation D869A abolishes both the catalytic activity of hTERT and its ability to extend cellular life span, demonstrating that the immortalizing capabilities of the enzyme are dependent on active catalysis. A second mutant of hTERT was examined that contains three copies of an HA epitope inserted at the C-terminus. This mutant produced telomerase activity in fibroblasts that was virtually indistinguishable from that of wild type telomerase when assayed in vitro. However, the forced expression of this mutant failed to maintain telomeres or extend cellular life span. Our results show that the catalytic activity of hTERT is required for cellular immortalization but that the presence of active telomerase does not necessarily imply telomere maintenance and immortality.

Base Sequence↗
PubMed1999

Role of telomerase in cellular proliferation and cancer.

Telomerase is a cellular reverse transcriptase that helps to provide genomic stability in highly proliferative normal, immortal, and tumor cells by maintaining the integrity of the chromosome ends, the telomeres. The activity of telomerase is associated with the majority of malignant human cancers. Telomerase or another mechanism for telomere maintenance is required for continuous tumor cell proliferation. Telomerase-positive cells that exit the cell cycle via quiescence downregulate telomerase through a transcriptional repression pathway. In the case of cell cycle exit via terminal differentiation, proteolysis of telomerase may also be involved. In response to mitogenic or growth factor signaling, telomerase-competent quiescent cells reenter the cell cycle and express telomerase activity independent of DNA synthesis. Under normal growth conditions, inhibition of telomerase activity in tumor-derived cells results in continued cell division coupled with telomere shortening, eventually followed by cellular senescence or death. Thus, repression of telomerase activity may be a novel adjuvant therapy for the treatment of human cancer and detection of telomerase activity may be important for cancer diagnostics.

Animals↗
PubMed1999

Absence of cancer-associated changes in human fibroblasts immortalized with telomerase.

The ectopic expression of telomerase in normal human cells results in an extended lifespan, indicating that telomere shortening regulates the timing of cellular senescence. As telomerase expression is a hallmark of cancer, we investigated the long-term effects of forced expression of human telomerase catalytic component (hTERT) in normal human fibroblasts. In vitro growth requirements, cell-cycle checkpoints and karyotypic stability in telomerase-expressing cells are similar to those of untransfected controls. In addition, co-expression of telomerase, the viral oncoproteins HPV16 E6/E7 (which inactivate p53 and pRB) and oncogenic HRAS does not result in growth in soft agar. Thus, although ectopic expression of telomerase in human fibroblasts is sufficient for immortalization, it does not result in changes typically associated with malignant transformation.

Catalytic Domain↗
PubMed1999

Progressive increase in telomerase activity from benign melanocytic conditions to malignant melanoma.

The expression of telomerase activity and the in situ localization of the human telomerase RNA component (hTR) in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Polymerase Chain Reaction-based assay (TRAP). High relative mean telomerase activity levels were detected in metastatic melanoma (subcutaneous metastases = 54.5, lymph node metastases = 56.5). Much lower levels were detected in primary melanomas, which increased with advancing levels of tumor cell penetration (Clark II = 0.02, Clark III = 1.1, and Clark IV = 1.9). Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.

Humans↗
PubMed1999

Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells.

Animals↗
PubMed1999

Both transcriptional and posttranscriptional mechanisms regulate human telomerase template RNA levels.

The human telomerase RNA component (hTR) is present in normal somatic cells at lower levels than in cancer-derived cell lines. To understand the mechanisms regulating hTR levels in different cell types, we have compared the steady-state hTR levels in three groups of cells: (i) normal telomerase-negative human diploid cells; (ii) normal cells transfected with the human telomerase catalytic subunit, hTERT; and (iii) cells immortalized in vitro and cancer cells expressing their own endogenous hTERT. To account for the differences in steady-state hTR levels observed in these cell types, we compared the transcription rate and half-life of hTR in a subset of these cells. The half-life of hTR in telomerase-negative cells is about 5 days and is increased 1.6-fold in the presence of hTERT. The transcription rate of hTR is essentially unchanged in cells expressing exogenous hTERT, and the increased steady-state hTR level appears to be due to the increased half-life. However, the transcription rate of hTR is greatly increased in cells expressing endogenous hTERT, suggesting some overlap in transcriptional regulatory control. We conclude that the higher hTR level in cells expressing an endogenous telomerase can be a result of both increased transcription and a longer half-life and that the longer half-life might be partially a result of protection or stabilization by the telomerase catalytic subunit. The 4-week half-life of hTR in H1299 tumor cells is the longest half-life yet reported for any RNA.

Animals↗
PubMed1999

Two inactive fragments of the integral RNA cooperate to assemble active telomerase with the human protein catalytic subunit (hTERT) in vitro.

We have mapped the 5' and 3' boundaries of the region of the human telomerase RNA (hTR) that is required to produce activity with the human protein catalytic subunit (hTERT) by using in vitro assembly systems derived from rabbit reticulocyte lysates and human cell extracts. The region spanning nucleotides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made with full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of active telomerase in vitro; this sequence encompasses a substantially larger portion of the 3' end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +325) that cannot produce telomerase activity when combined separately with hTERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vitro.

Animals↗
PubMed1999

Detection of telomerase activity in exfoliated cancer cells obtained from bile.

Telomerase is detected by the telomeric repeat amplification protocol (TRAP) assay in more than 85% of primary cancers. In the present study, we determined telomerase activity using exfoliated bile cells obtained from biliary tract neoplasia specimens. The aim of this study was to provide additional information regarding minimally invasive approaches to the detection of biliary tract cancer in combination with routine cytologic examination. We analyzed for telomerase activity bile juice from patients with gallbladder carcinoma, cholangiocarcinoma, cholecystitis and cholangitis. Semiquantitative determination of telomerase activity was performed using both a fluorescence-based TRAP assay on cell extracts and at the cellular level by an in situ TRAP assay. The fluorescence-based TRAP assay detected bile telomerase activity in samples from 4 of 10 patients with biliary tract cancer. In contrast, the in situ TRAP assay detected telomerase positive cells in samples from 6 of 10 patients with biliary tract cancer. However, only one of these samples showed class V cytology. A combination of semiquantitative analysis and an in situ TRAP assay to detect telomerase positive cells may improve the diagnosis of biliary tract cancers with the combination of routine cytologic examination.

Adult↗
PubMed1999

Rapid detection of MYCN gene amplification and telomerase expression in neuroblastoma.

Amplification of the MYCN gene and high telomerase activity predict a poor prognosis for the patients with neuroblastoma. We used PCR techniques for rapid detection of MYCN gene amplification and human telomerase reverse transcriptase (hTERT) expression in neuroblastoma specimens. The detection of MYCN gene amplification is based on differential PCR in which three primer pairs were used to coamplify a 178-bp fragment of target MYCN gene with two reference gene fragments, a 237-bp of p53 exon 7 and a 120-bp of beta-globin exon 3, in a single tube of 40 surgically resected tumor samples. MYCN amplification was identified by this differential PCR in all 10 samples carrying more than 10 copies (already known to have MYCN gene amplification by Southern blot analysis). There were no false-negative or false-positive cases, and the relative intensity of MYCN bands in the differential PCR correlated significantly with the copy number determined by Southern blot analysis (y = 0.99, P<0.0001). This protocol was also applicable in the biopsy or aspirated samples, as well as the paraffin-embedded tissues, and in detecting intratumoral heterogeneity. Using RT-PCR procedures, hTERT mRNA expression was detectable in all 13 tumors with high telomerase activity. These nonradioisotopic PCR-based protocols for detecting MYCN gene amplification and hTERT mRNA expression are rapid and reliable and are likely to be useful to determine the biological behavior of neuroblastoma.

Biomarkers, Tumor↗
PubMed1999

Telomere stability is frequently impaired in high-risk groups of patients with myelodysplastic syndromes.

Genomic instability induces an accumulation of genetic changes and may play a role in the pathogenesis of myelodysplastic syndromes (MDS). To clarify the possible association between genomic instability and clinical outcome in MDS patients, we compared telomere dynamics to the recently established International Prognostic Scoring System (IPSS) risk groups for MDS. We measured the terminal restriction fragments (TRFs) of 93 patients with MDS at the time of diagnosis, and telomerase activity was analyzed in 62 patients with MDS using the PCR-based telomeric repeat amplification protocol (TRAP) assay. A total of 53 of 93 MDS patients had TRFs within the age-matched normal range, and the remaining patients showed shortened TRFs (35 patients) or elongated TRFs (5 patients). MDS patients with shortened TRFs had a significantly low hemoglobin concentration (P = 0.04), a high percentage of marrow blasts (P = 0.02), and a high incidence of cytogenetic abnormalities (P < 0.05). The incidence of leukemic transformation was significantly high in patients with shortened TRF length (P < 0.05). In addition, patients with shortened TRF length were frequently seen in the IPSS high-risk group (P < 0.01). Most of the MDS patients had normal-to-low levels of telomerase activity, suggesting that changes in TRF length rather than telomerase activity may more accurately reflect the pathophysiology of MDS. MDS patients with shortened TRF length had a very poor prognosis (P < 0.01), suggesting that telomere dynamics may be linked to clinical outcome in MDS patients. Thus, an abnormal mechanism of telomere maintenance in subgroups of MDS patients may be an early indication of genomic instability. This study demonstrates that telomere stability is frequently impaired in a high-risk group of MDS patients and suggests that, in combination with the IPSS classification system, measurement of TRFs may be useful in the future to stratify MDS patients according to risk and manage the care of MDS patients.

Acute Disease↗
PubMed1998

Characterization of paired tumor and non-tumor cell lines established from patients with breast cancer.

The goal of our study was to develop a panel of tumor cell lines along with paired non-malignant cell lines or strains collected from breast cancers, predominantly primary tumors. From a total of 189 breast tumor samples consisting of 177 primary tumors and 12 metastatic tissues, we established 21 human breast tumor cell lines that included 18 cell lines derived from primary tumors and 3 derived from metastatic lesions. Cell lines included those from patients with germline BRCA1 and FHIT gene mutations and others with possible genetic predisposition. For 19 tumor cell lines, we also established one or more corresponding non-malignant cell strains or B lymphoblastoid (BL) lines, which included 16 BL lines and 7 breast epithelial (2) or stromal (5) cell strains. The present report describes clinical, pathological and molecular information regarding the normal and tumor tissue sources along with relevant personal information and familial medical history. Analysis of the breast tumor cell lines indicated that most of the cell lines had the following features: they were derived from large tumors with or without axillary node metastases; were aneuploid and exhibited a moderate to poorly differentiated phenotype; were estrogen receptor (ER)- and progesterone receptor (PR)-negative; and overexpressed p53 and HER2/neu proteins. Of 13 patients with primary breast cancers receiving curative intent mastectomies, 7 were dead after a mean period of 10 months. Our panel of paired tumor and non-malignant cell lines should provide important new reagents for breast cancer research.

Adult↗
PubMed1998

In situ hybridization for the detection of telomerase RNA in the progression from Barrett's esophagus to esophageal adenocarcinoma.

BACKGROUND: The rate of incidence of esophageal adenocarcinoma has surpassed that of all other malignancies in the U.S. Esophageal adenocarcinoma arises from Barrett's esophagus through the multistep accumulation of genetic mutations in metaplastic cells, accompanied by histologic changes from metaplasia to low grade and high grade dysplasia and finally to carcinoma. Although telomerase activity has been demonstrated in 85-90% of human cancers, to the authors' knowledge there are no reports regarding esophageal adenocarcinoma. The objective of this study was to determine whether telomerase RNA is increased in esophageal adenocarcinoma and at what point in the histologic progression such an increase occurs. METHODS: Formalin-fixed, paraffin-embedded esophageal biopsies from patients with Barrett's esophagus and surgical resection specimens of esophageal adenocarcinoma containing varying stages of neoplastic progression were obtained. Adjacent sections from each specimen were evaluated by routine histology and in situ hybridization for the RNA component of human telomerase. RESULTS: The authors found that 100% of esophageal adenocarcinomas and high grade dysplasias were strongly positive for telomerase RNA. Basal crypt cells of Barrett's metaplasia demonstrated weak to moderate telomerase RNA in 70% of cases, whereas 90% of low grade dysplasias had moderate levels of telomerase RNA. CONCLUSIONS: The results of this study showed that 1) Barrett's epithelium may contain a population of immortalized cells, 2) a marked increase in the level of telomerase RNA accompanies the transition from low grade to high grade dysplasia, and 3) high levels of telomerase RNA accompany the development of esophageal adenocarcinoma in the vast majority of cases.

Adenocarcinoma↗
PubMed1998

Telomerase activity during spontaneous immortalization of Li-Fraumeni syndrome skin fibroblasts.

Li-Fraumeni Syndrome (LFS) is characterized by heterozygous germline mutations in the p53 gene. Accompanied by genomic instability and loss or mutation of the remaining wild type p53 allele, a low frequency of spontaneous immortalization in LFS fibroblasts occurs. It is believed that the loss of p53 wild type function contributes to immortalization of these LFS fibroblasts, but it is not clear if this is sufficient. Because stabilization of telomere length is also thought to be a necessary step in immortalization, telomerase activity, expression of the telomerase RNA component (hTR) and telomere length were anlaysed at various passages during the spontaneous immortalization of LFS skin fibroblasts. One LFS strain which immortalized, MDAH087 (087), had no detectable telomerase activity whereas another LFS strain which immortalized, MDAH041 (041), had detectable telomerase activity. In preimmortal cells from both strains, hTR was not detected by in situ hybridization. Immortal 087 cells remained negative for hTR, while immortal 041 cells demonstrated strong hTR in situ hybridization signals. 087 cells had long and heterogenous telomeres whereas telomeres of 041 cells had short, stable telomere lengths. Tumorigenicity studies in nude mice with ras-transformed 087 and 041 cells resulted in both cell lines giving rise to tumors and retaining telomerase status. Overall these results suggest that strain specificity may be important in telomerase re-activation and that both abrogation of p53 function and a mechanism to maintain telomeres are necessary for immortalization.

Animals↗
PubMed1998

Characterization of a breast cancer cell line derived from a germ-line BRCA1 mutation carrier.

A tumor cell line, HCC1937, was established from a primary breast carcinoma from a 24-year-old patient with a germ-line BRCA1 mutation. A corresponding B-lymphoblastoid cell line was established from the patient's peripheral blood lymphocytes. BRCA1 analysis revealed that the tumor cell line is homozygous for the BRCA1 5382insC mutation, whereas the patient's lymphocyte DNA is heterozygous for the same mutation, as are at least two other family members' lymphocyte DNA. The tumor cell line is marked by multiple additional genetic changes including a high degree of aneuploidy, an acquired mutation of TP53 with wild-type allele loss, an acquired homozygous deletion of the PTEN gene, and loss of heterozygosity at multiple loci known to be involved in the pathogenesis of breast cancer. Comparison of the primary tumor with the cell line revealed the same BRCA1 mutation and an identical pattern of allele loss at multiple loci, indicating that the cell line had maintained many of the properties of the original tumor. This breast tumor-derived cell line may provide a useful model system for the study of familial breast cancer pathogenesis and for elucidating BRCA1 function and localization.

Adult↗
PubMed1998

Telomerase activity in lung cancer cells obtained from bronchial washings.

BACKGROUND: Telomerase, a ribonucleoprotein enzyme that functions in the maintenance of telomeres (specialized structures at the ends of chromosomes), has been reported to be a novel diagnostic marker for malignant diseases. We sought to determine whether measurement of telomerase activity in bronchial washings is of value in the diagnosis of lung cancer. METHODS: Extracts of cells in bronchial washings were analyzed for telomerase activity by use of a telomeric repeat amplification protocol (TRAP) assay. Telomerase activity inside cells was evaluated by use of an in situ TRAP assay. The results of both TRAP assays were compared with those obtained from cytologic examination, which employed standard Papanicolaou staining. RESULTS: When results from the two TRAP assays were combined, telomerase activity was detected in bronchial washings from 18 (82%; 95% confidence interval [CI] = 60%-95%) of 22 patients with lung cancer. In contrast, cancer cells were detected by cytologic examination in the bronchial washings of nine (41%; 95% CI = 21%-64%) of the same 22 patients, a statistically significant difference (two-sided P = .0061). In patients with lung cancer, telomerase-positive cells could be detected in bronchial washings irrespective of tumor location--11 of 14 (79%; 95% CI = 49%-95%) peripheral cancerous lesions and seven of eight (88%; 95% CI = 47%-100%) central cancerous lesions were detected by use of TRAP assays (for comparison, two-sided P = .5349). CONCLUSIONS: A high percentage of patients with lung cancers had detectable telomerase activity in bronchial washings. Thus, the use of a cell extract-based or an in situ TRAP assay in addition to cytologic examination may make the diagnosis of lung cancer more reliable.

Adult↗