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J W Shay

Publications and source records attributed to J W Shay.

At least 73 records · Page 4Linked to original sources
PubMed1998

Telomerase activity and expression of its RNA component in cervical lesions.

BACKGROUND: The authors investigated telomerase enzyme activity and expression of its RNA component (hTR) during the multistage pathogenesis of cervical carcinomas, and correlated activation with histopathologic findings and human papillomavirus (HPV) infection. METHODS: The authors analyzed 180 cervical specimens for enzyme activity, and analyzed hTR expression in an additional 55 samples from archival carcinoma cases. Polymerase chain reaction-based assays were used to determine telomerase enzyme activity and HPV infection, whereas a radioactive in situ assay was used for hTR expression. RESULTS: Telomerase enzyme activity was present in some samples of histologically normal epithelium (18 of 138; 13%) and low grade squamous intraepithelial lesions (LSIL) (7 of 21; 33%), and in most high grade squamous intraepithelial lesions (HSIL) (13 of 21; 62%). The relative levels of telomerase activity were low in all preinvasive specimens except for three samples of HSIL with high activity. Although 21% of the brush samples had evidence of HPV infection, there was no obvious correlation between telomerase activity and HPV status. hTR expression was low in normal squamous/glandular epithelium and LSIL lesions, in which it was limited to the basal cells. In squamous and glandular in situ and invasive carcinomas, increased and dysregulated hTR expression was observed, although heterogeneity was noted. Intense focal up-regulation of hTR expression occurred in a subset of in situ lesions. CONCLUSIONS: Increased frequency and dysregulation of telomerase activation is correlated with increasing severity of histopathologic changes, but not with HPV infection. Whether dysregulated activity is a prognostic marker for development of invasive carcinoma remains to be determined.

Adenocarcinoma↗
PubMed1998

Extension of life-span by introduction of telomerase into normal human cells.

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

Biomarkers↗
PubMed1998

Detection of telomerase activity in breast masses by fine-needle aspiration.

BACKGROUND: Telomerase is an RNA-dependent DNA polymerase that compensates for the telomere shortening that occurs in its absence. Reactivation of telomerase is thought to be an important step in cellular immortalization, and recent studies have indicated that telomerase activity is often detected in primary human malignancies. The clinical implications of telomerase activity in human tumors are currently under investigation. METHODS: Eighty-nine samples (46 FNAs and 43 gross tissue biopsies) from 44 patients with breast masses were analyzed prospectively for the presence of telomerase activity by a modification of the telomere repeat amplification protocol (TRAP). All samples were obtained directly from the excised mass at the time of specimen removal in the operating room. RESULTS: Telomerase activity was detected in 17 of 19 (90%) FNA samples and 15 of 18 (83%) invasive breast cancer tissue biopsies. Telomerase was also detected in 9 of 16 (56%) FNAs and 8 of 15 (53%) tissue biopsies from 16 fibroadenomas. Other benign proliferative lesions (n = 5) did not have detectable telomerase activity in either FNA or tissue specimens. FNA-TRAP results correlated with the gross tissue specimen TRAP results in 95% of all cases. CONCLUSION: The FNA-TRAP assay for telomerase detection is a highly sensitive and accurate method for the detection of telomerase activity in breast masses. Future application of these techniques should facilitate evaluation of telomerase as a tumor marker in the clinical management of breast and other solid malignancies.

Adolescent↗
PubMed1998

Telomeric length and telomerase activity vary with age in peripheral blood cells obtained from normal individuals.

The telomerase activity and length of telomeres of peripheral blood mononuclear cells obtained from 124 healthy individuals aged 4-95 years was measured. Telomerase activity level was semiquantitatively assessed by a fluorescent-telomeric repeat amplification protocol (fluorescent-TRAP) using an internal telomerase assay standard, fluorescent primers and an automated laser fluorescent DNA sequencer. Telomeric length, measured by assay of terminal restriction fragments (TRFs), was determined in HinfI-digested DNA by Southern blot analysis using a (TTAGGG)4 probe. TRF length was determined in 80 individuals and age-related progressive reduction of size was observed. TRF length in peripheral blood mononuclear cells obtained from normal individuals (aged 4-39 years) decreased by approximately 84 bp per year, while in individuals aged > or = 40 years it decreased by 41 bp per year. In contrast, telomerase activity showed an apparent biphasic pattern with aging. Individuals aged 4-39 years showed a progressive decrease in telomerase activity, whereas 65% of those aged > or = 40 years showed relatively stable but very low telomerase activity, and the remaining individuals aged > or = 40 years had no detectable telomerase activity. These data obtained from normal individuals might in the future be of value to help risk stratify and manage the care of patients with leukemia.

Adolescent↗
PubMed1998

In situ hybridization for telomerase RNA in routine cytologic brushings for the diagnosis of pancreaticobiliary malignancies.

BACKGROUND: Brush cytology for the diagnosis of pancreaticobiliary malignancy has an overall sensitivity of 50%. Accurate and specific markers are therefore needed for the optimal evaluation of pancreaticobiliary strictures. Telomerase activity is present in 85% to 90% of all human cancers. We sought to determine the utility of in situ hybridization for telomerase RNA in endoscopic brushings for the diagnosis of pancreaticobiliary malignancy. METHODS: Endoscopic brushings from 18 patients with pancreatic or biliary strictures were evaluated by routine cytology and in situ hybridization for telomerase RNA. RESULTS: Eight of 18 strictures were malignant. Cytology was positive in 5 patients, whereas telomerase RNA was positive in 6. All malignancies were diagnosed by either cytology or telomerase RNA; however, both studies were positive in only 3. There were no false-positive results by either technique. CONCLUSION: The detection of telomerase RNA in endoscopic brushings may be an important adjunct to cytology for cancer diagnosis in pancreaticobiliary strictures.

Adult↗
PubMed1998

Expression of the RNA component of telomerase in Wilms tumor and nephrogenic rest recapitulates renal embryogenesis.

Telomerase is a ribonucleoprotein enzyme associated with cellular immortality and has been detected in the vast majority of adult tumors. Wilms tumor is a histologically diverse embryonal malignancy of childhood, and the histological features of Wilms tumor and its precursor lesion, the nephrogenic rest, recapitulate the components of normal renal embryogenesis. Both the epithelial and the stromal components of Wilms tumor arise by differentiation of primitive mesodermal blastema. We compared expression of the RNA component of human telomerase (hTR) in normal developing kidneys, Wilms tumors, and nephrogenic rests and correlated expression of hTR with cell proliferation. Using a radioactive in situ hybridization method, we examined archival material from 17 Wilms tumors (including nine with nephrogenic rests), four therapeutically aborted embryos (37 to 56 days), three fetuses on whom autopsies had been performed, and one neonate for expression of hTR. Proliferative index was measured by immunohistochemical staining for MIB1. In the embryonic kidney, Wilms tumors, and nephrogenic rests, the patterns of hTR expression were similar: expression was usually maximal within the immature epithelial elements followed by the poorly differentiated blastema, but was weak or absent in the immature stroma. Mature tubules, glomeruli, and stroma were negative for hTR expression, as were differentiated heterologous elements present in post-therapy Wilms tumors. There was only a partial relationship between proliferative index and hTR expression. In the embryonic kidney, Wilms tumors, and nephrogenic rests, blastema had the highest proliferative index, whereas the indices were significantly lower in the immature epithelium and stroma. The proliferative index in mature and heterologous elements was low or zero. Thus, the pattern of hTR expression in Wilms tumor and its precursor lesion recapitulates embryogenesis precisely and may represent that aspect of the persistent fetal phenotype which predisposes to the development of malignancy.

Child, Preschool↗
PubMed1998

In situ hybridization analysis of the expression of human telomerase RNA in normal and pathologic conditions of the skin.

Human telomerase RNA (hTER) expression in skin was examined by in situ hybridization analysis. All newborn foreskins examined (n = 5) expressed hTER in epidermal basal cells at moderate levels. Telomerase RNA was not detectable in most adult specimens from sun protected areas (six of seven), whereas all samples obtained from sun exposed areas (n = 8) showed moderate hTER signals in epidermal basal cells. Telomerase RNA was also detected at moderate to strong levels in basal cells of psoriasis, contact dermatitis, and the proliferative cells of the anagen hair bulb. Basal cell carcinoma samples (14 of 15) had moderate to high hTER expression throughout the entire tumor, whereas squamous cell carcinomas (seven of eight) showed variable intensities of hTER expression but only in the cells at the periphery of tumor nests. All melanomas examined (n = 5) had moderate hTER expression in all tumor cells. The hTER signal intensities in skin tumors did not correlate with the age or sex of the donors, the clinical history of the lesions, or the histologic subtypes. To address whether hTER expression correlated with the proliferative state, sequential sections were stained with anti-Ki-67 antibody, a proliferation marker. In newborn foreskins, squamous cell carcinomas, and basal cell carcinomas, the distributions of hTER and Ki-67 were similar but not always identical. Telomerase RNA was more abundant than Ki-67 in the basal and suprabasal layer of newborn foreskins, suggesting that hTER expression is present both in actively cycling and in resting cells.

Biomarkers↗
PubMed1998

Enhanced detection of human telomerase activity.

The detectability of telomerase activity in human cells almost always correlates with indefinite proliferation capability (immortalization). To make quantitative statements about telomerase activity levels, complete extraction of the telomerase activity is needed. A series of detergents was tested for this purpose, and a combination of NP-40 and sodium deoxycholate (NaDOC) was found to be the most efficient for extracting telomerase activity. Tumor-derived cell lines originally thought to contain differing amounts of telomerase on the basis of the original CHAPS bases extraction procedures have nearly equivalent amounts of activity when extracted with the NP-40/NaDOC lysis buffer. These results indicate that these lysis conditions can be used to extract telomerase activity more efficiently from tumor-derived cell lines.

Biochemistry↗
PubMed1998

Telomerase activity in rheumatoid synovium correlates with the mononuclear cell infiltration level and disease aggressiveness of rheumatoid arthritis.

OBJECTIVE: To determine whether the presence of telomerase activity in synovial tissues could provide insights into the pathogenesis of rheumatoid arthritis (RA). METHODS: Synovial tissue samples obtained from patients with RA or osteoarthritis (OA) were examined for telomerase activity using the telomeric repeat amplification protocol (TRAP). RESULTS: Telomerase activity was detected in over half the RA synovial tissues (14/25, 56%) but in no OA synovial tissue samples (0/15). Telomerase activity was detected in the mononuclear cells isolated from telomerase-positive RA synovial tissue, but not in cultured adherent cells. In RA synovial tissue with positive telomerase activity mononuclear cell infiltration levels were increased (p < 0.001). In addition, patients with RA with positive telomerase activity had undergone joint operations at an earlier age (p = 0.030) and more often (p = 0.023) compared to those without telomerase activity. CONCLUSION: Telomerase activity in RA synovial tissue is likely derived from infiltrating mononuclear cells and may be involved in the pathogenesis of RA. Clinically, the presence of telomerase activity in RA may be an indicator of a more aggressive phenotype.

Aged↗
PubMed1998

Analysis of telomere lengths in human corneal endothelial cells from donors of different ages.

PURPOSE: To investigate the telomere hypothesis of cellular aging as the mechanism for cell cycle arrest in normal human corneal endothelium. METHODS: The corneal endothelium and epithelium from 21 human corneas from 13 donors 5 weeks to 84 years of age were dissected and frozen at -70 degrees C. Purified DNA, digested with the restriction enzyme, HinfI, was run on 0.7% agarose gels, probed with radiolabeled (AATCCC)4, and exposed to a phosphor screen. The length of the terminal restriction fragment (TRF) was determined by densitometry. RESULTS: The cells of the corneal endothelium had TRF lengths ranging from 11.0 to 14.0 kbp (mean, 12.2 +/- 0.9). Corneal epithelial specimens showed TRF lengths that were always less than (mean, 10.4 +/- 1.0; range 9.0-12.0) the corresponding endothelial TRF lengths. Human corneal endothelial cells, transformed with human papillomavirus type 16 oncogenes E6 and E7, showed decreasing TRF lengths from 11 kbp at population doubling level (PDL) 15 to 9.5 kbp at PDL 73. Neither the endothelial and epithelial cells from human donors nor the transformed pre-immortalized human endothelial cells showed evidence of telomerase activity. CONCLUSIONS: Human corneal endothelial cells have long telomeres throughout life. Their limited replicative ability does not appear to result from critically short telomere lengths.

Adolescent↗
PubMed1998

Telomerase enzyme activity and RNA expression during the multistage pathogenesis of breast carcinoma.

Telomerase, an RNA-containing enzyme, is associated with cellular immortality and malignancy. We investigated the role of telomerase during the multistage pathogenesis of breast cancer. We used the semiquantitative, PCR-based telomeric repeat amplification protocol assay for enzyme activity (42 specimens from 42 patients) and a radioactive in situ assay for expression of its RNA component (human telomerase RNA; hTR) for the identification of telomerase-positive cells in archival resection samples (n = 67 from 39 patients). Low telomerase activity was detected in 1 (14%) of 7 samples of benign breast disease, in 4 (67%) of 6 fibroadenomas, in 11 (92%) of 12 carcinoma in situ (CIS) lesions, and in 16 (94%) of 17 invasive breast cancers. There was a progressive increase in the mean telomerase levels with progressive increase in severity of histopathological change (P < 0.05). Almost all of 67 resection samples expressed hTR, irrespective of histology. Expression was low to moderate in some samples of normal epithelium and nonproliferative fibrocystic changes. hTR expression was limited to epithelial cells; expression in stromal cells, including those in fibroadenomas, was negative. Increased hTR expression was observed in some foci of apocrine metaplasia and atypical hyperplasia. Increased hTR expression was also observed in all CIS and invasive lesions, although considerable heterogeneity was noted. Focal up-regulation was frequently noted in CIS lesions in the vicinity of invasive tumors. Thus, up-regulation of hTR may be a predictive marker for invasive tumor development.

Breast↗
PubMed1998

Comparison of human telomerase RNA and telomerase activity in urine for diagnosis of bladder cancer.

New, noninvasive methods for the early detection of urothelial carcinomas of the urinary bladder are needed for the diagnosis, follow-up, and screening of patients with bladder cancer. Detection of the enzyme telomerase in urine could offer these new diagnostic possibilities. The standard technique for detecting telomerase activity is the telomeric repeat amplification protocol (TRAP assay). Because of the instability of the ribonucleoprotein telomerase in an aggressive medium, such as urine, investigations conducted to date have yielded nonuniform or even contradictory findings. This study compares the detection of human telomerase RNA (hTR) by reverse transcriptase-PCR (RT-PCR) with detection of telomerase activity by the TRAP assay in the diagnosis of urothelial carcinoma of the urinary bladder. Sedimented cells obtained from urine of 30 patients with urothelial carcinoma, 15 patients with benign urological disorders, 3 patients as part of follow-up for malignant disease, and 20 healthy subjects were examined for the presence of hTR and for telomerase activity (TRAP). In patients with bladder cancer, telomerase activity was detected by the TRAP assay in only 2 of 30 specimens (7%). However, increased levels of hTR were detected by RT-PCR in 25 of the same 30 cases (83%). For patients with benign urological disorders, such as urolithiasis or urinary tract infections, hTR was detected in samples obtained from 4 of 15 patients (27%). Low hTR expression levels were found in 15% of the healthy controls. The detection of hTR by RT-PCR represents a promising new method for detecting malignant cells in urine.

Humans↗
PubMed1998

Expression of the RNA component of telomerase during human development and differentiation.

We used a radioactive in situ method to study expression of the RNA component of human telomerase (hTR) during normal human development and differentiation using archival tissues. In embryonic tissues, the highest and most uniform expression was present in undifferentiated neuroepithelium. Expression was stronger in immature epithelium than in accompanying immature mesenchyme. Differentiation of most tissues was accompanied by decreased or absent expression. Except for testis and adrenal, the adult pattern of expression was present by the 10th postnatal week. In adult tissues, high expression was present in the testis (primary spermatocytes and Sertoli cells), moderate expression was present in lymphoid follicles (germinal centers), and weak expression was present in epithelia (regenerative cells) but was absent in the nervous system and mesenchymal derived tissues. Expression in adult tissues was predominantly limited to dividing cells, although certain differentiated postmitotic cells expressed the hTR. Our studies demonstrate the complex interrelationship of hTR expression with human development, differentiation, and cell division.

Adolescent↗
PubMed1998

Telomerase activity as a novel marker of lung cancer and immune-associated lung diseases.

Telomerase maintains telomere length and is considered to be necessary for the indefinite proliferation of human cells. Telomerase activity is detected not only in germline and immortal cancer cells, but also in stem/progenitor cells of renewal tissues and activated lymphocytes. While it is generally agreed that telomerase is a useful tumor marker, the utility of telomerase activity in non-cancerous cells should also be considered. In the present study, we quantitatively examined telomerase activity in 56 cytology samples and 106 bronchoalveolar lavage samples obtained from patients with various respiratory diseases. Fourteen of 34 samples obtained from lung cancer patients showed detectable telomerase activity, while only 7 of 128 samples obtained from patients without lung cancer did (p<0.001). Moreover, 12 of 14 telomerase-positive samples with lung cancer showed strong signals, while none without lung cancer did. Among 106 non-cancerous bronchoalveolar lavage samples, 4 telomerase positive samples had increased number of lymphocytes and increased disease progression. These findings indicate that evaluation of telomerase activity may not only be a useful diagnostic test for lung cancer, but may also be a marker of disease aggressiveness for immune-associated lung diseases.

Biomarkers↗
PubMed1998

Characterization of a soluble KGF receptor cDNA from human corneal and breast epithelial cells.

PURPOSE: Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family FGF-7. It exhibits potent mitogenic activity for epithelial cells, including corneal and mammary epithelial cells. A messenger RNA has been reported that is generated by alternative splicing of bek that putatively codes only for the extracellular ligand-binding domain of KGF receptor (soluble KGF receptor). In the present study, the expression of the mRNA coding for this alternative bek transcript was examined and the corresponding protein characterized. METHODS: Alternative messenger RNA transcripts were detected in various cell lines or tissues using reverse transcription-polymerase chain reaction (RT-PCR) and RNase protection assay. NIH/3T3 fibroblast cells and 293 kidney embryonic epithelial cells were stably transfected with soluble KGF receptor cDNA and transmembrane KGF receptor cDNA. Soluble KGF receptor protein was produced using a baculovirus-insect expression system. Soluble KGF receptor protein was detected using western and dot blot analyses. Binding assays and cross-linking labeling were used to determine the affinity and specificity of soluble KGF receptor. A mitogenic assay was performed to examine the function of the soluble KGF receptor. RESULTS: The soluble KGF receptor mRNA was primarily expressed in epithelial cells, including cells from the cornea and breast. Cross-linking labeling and affinity-binding assays with 125I-KGF showed that the soluble KGF receptor bound KGF (FGF-7) but not FGF-1 or FGF-2. Soluble KGF receptor was detected in the culture medium of cells stably transfected with soluble KGF receptor cDNA but not with transmembrane KGF receptor cDNA, suggesting that the soluble receptor was generated by mRNA splicing and probably not by proteolysis or posttranslational processing. Soluble KGF receptor inhibited KGF binding to transmembrane KGF receptor and DNA synthesis in BALB/MK epidermal keratinocytes in response to KGF, suggesting that soluble KGF receptor expression could provide a mechanism for the cell to downregulate responses to KGF. CONCLUSIONS: A truncated soluble KGF receptor expressed in corneal and other epithelial cells probably functions to downregulate the response of the cell to KGF.

3T3 Cells↗