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Biomedical subjects

C B Harley

Publications and source records attributed to C B Harley.

At least 37 records · Page 2Linked to original sources
PubMed1995

Telomerase activity in normal leukocytes and in hematologic malignancies.

Telomeres are essential for function and stability of eukaryotic chromosomes. In the absence of telomerase, the enzyme that synthesizes telomeric DNA, telomeres shorten with cell division, a process thought to contribute to cell senescence and the proliferative crisis of transformed cells. We reported telomere stabilization concomitant with detection of telomerase activity in cells immortalized in vitro and in ovarian carcinoma cells, and suggested that telomerase is essential for unlimited cell proliferation. We have now examined the temporal pattern of telomerase expression in selected hematologic malignancies. We found that, unlike other somatic tissues, peripheral, cord blood, and bone marrow leukocytes from normal donors expressed low levels of telomerase activity. In leukocytes from chronic lymphocytic leukemia (CLL) patients, activity was lower than in controls in early disease, and comparable with controls in late disease. Relative to bone marrow, telomerase activity was enhanced in myelodysplastic syndrome (MDS) and more significantly so in acute myeloid leukemia (AML). Regardless of telomerase levels, telomeres shortened with progression of the diseases. Our results suggest that early CLL and MDS cells lack an efficient mechanism of telomere maintenance and that telomerase is activated late in the progression of these cancers, presumably when critical telomere loss generates selective pressure for cell immortality.

Acute Disease↗
PubMed1995

Evidence for a critical telomere length in senescent human fibroblasts.

Telomeres, the G/C-rich DNA sequences capping the ends of all eukaryotic chromosomes, have been shown to shorten during replicative aging of normal cells in vitro and in vivo. Moreover, variation in the initial length of terminal restriction fragments (TRF) accounts for much of the variation in replicative capacity of fibroblast cultures from different donors. Since replicative capacity also varies significantly between clones in a mass culture of fibroblasts from a single donor, we wished to further test the hypothesis that the shortening of telomeres to a critical or threshold length acts as a signal for cell senescence. Thus, we measured TRF length and total telomeric signal intensity for 35 clonal fibroblast populations at early passage and at senescence. Replicative capacity was found to be directly proportional to mean TRF length (m = 7.2 population doublings/kbp, r = 0.65, P = 0.0004) and total signal intensity (m = 25.0 population doublings/unit, r = 0.63, P < 0.003) at early passage. More importantly, the variability in both mean TRF length and signal intensity (F = 2.0 and 2.9; P = 0.02 and 0.03, respectively) at senescence was markedly less than that at early passage. Although initial telomere length cannot account for all of the interclonal variability in replicative capacity, our observations support the existence of a critical telomere length in senescing cells and a causal role of telomere shortening in cell senescence.

Adult↗
PubMed1995

Telomere shortening is associated with cell division in vitro and in vivo.

In humans, the amount of terminal (TTAGGG)n, telomeric DNA decreases during aging of various somatic cell types in vitro and in vivo. While the factors accounting for telomere shortening have not been thoroughly established, the inability of the DNA replication machinery to completely copy chromosomal termini (the "end replication problem") and the absence in somatic cells of telomerase, the enzyme that synthesizes telomeric DNA de novo, is a likely mechanism. One prediction of this hypothesis is that telomere shortening should be dependent on cell division. Thus we analyzed telomere length in actively dividing and quiescent cells in vitro and in vivo. In circular outgrowths of cultured human diploid fibroblasts (HDF), cells at the outer periphery had a significantly lower mean terminal restriction fragment (TRF) length (P = 0.011) and telomeric signal intensity (P = 0.024) than cells at the center. Also, the rate of telomere shortening over time for HDFs held quiescent was not statistically significant (m = -12 bp/day, P = 0.16) while that for serially passaged cells was significant (m = -34 bp/day, P = 0.017). To examine the rate of telomere shortening for quiescent cells in vivo, we measured mean TRF length in brain tissue from adult donors ranging in age from 32-75 years. No significant decrease was observed as a function of donor age (P = 0.087), in contrast to the shortening of telomere length that occurs during in vivo aging of mitotically active cells (P = 0.0001). These observations show that telomere shortening is largely, if not entirely, dependent on cell division and support the end replication problem as a mechanism for this process and the use of telomere length as a biomarker for replicative capacity.

Adult↗
PubMed1995

Telomeres and telomerase in aging and cancer.

Telomeres are maintained by the novel ribonucleoprotein enzyme telomerase. Telomerase activity is repressed in most somatic human cells, leading to telomere loss during replicative aging in vivo and in vitro. However, telomerase appears to be reactivated in essentially all human cancers. With the recent cloning of the RNA component of telomerase from several species, the stage is now set for critical tests of the role of telomeres and telomerase in aging and cancer.

Aging↗
PubMed1994

Specific association of human telomerase activity with immortal cells and cancer.

Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100 immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated in cancer, where immortal cells are likely required to maintain tumor growth.

Base Sequence↗
PubMed1994

Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age.

The proliferative life-span of the stem cells that sustain hematopoiesis throughout life is not known. It has been proposed that the sequential loss of telomeric DNA from the ends of human chromosomes with each somatic cell division eventually reaches a critical point that triggers cellular senescence. We now show that candidate human stem cells with a CD34+CD38lo phenotype that were purified from adult bone marrow have shorter telomeres than cells from fetal liver or umbilical cord blood. We also found that cells produced in cytokine-supplemented cultures of purified precursor cells show a proliferation-associated loss of telomeric DNA. These findings strongly suggest that the proliferative potential of most, if not all, hematopoietic stem cells is limited and decreases with age, a concept that has widespread implications for models of normal and abnormal hematopoiesis as well as gene therapy.

Adolescent↗
PubMed1994

Telomerase activity in human ovarian carcinoma.

Telomeres fulfill the dual function of protecting eukaryotic chromosomes from illegitimate recombination and degradation and may aid in chromosome attachment to the nuclear membrane. We have previously shown that telomerase, the enzyme which synthesizes telomeric DNA, is not detected in normal somatic cells and that telomeres shorten with replicative age. In cells immortalized in vitro, activation of telomerase apparently stabilizes telomere length, preventing a critical destabilization of chromosomes, and cell proliferation continues even when telomeres are short. In vivo, telomeres of most tumors are shorter than telomeres of control tissues, suggesting an analogous role for the enzyme. To assess the relevance of telomerase and telomere stability in the development and progression of tumors, we have measured enzyme activity and telomere length in metastatic cells of epithelial ovarian carcinoma. We report that extremely short telomeres are maintained in these cells and that tumor cells, but not isogenic nonmalignant cells, express telomerase. Our findings suggest that progression of malignancy is ultimately dependent upon activation of telomerase and that telomerase inhibitors may be effective antitumor drugs.

Ascites↗
PubMed1994

Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes.

We have measured telomere length and telomerase activity throughout the life span of clones of human B lymphocytes transformed by Epstein-Barr virus. Shortening of telomeres occurred at similar rates in all populations and persisted until chromosomes had little telomeric DNA remaining. At this stage, some of the clones entered a proliferative crisis and died. Only clones in which telomeres were stabilized, apparently by activation of telomerase, continued to proliferate indefinitely, i.e., became immortal. Since loss of telomeres impairs chromosome function, and may thus affect cell survival, we propose that telomerase activity is required for immortality. We have now detected this enzyme in a variety of immortal human cells transformed by different viruses, indicating that telomerase activation may be a common step in immortalization.

B-Lymphocytes↗
PubMed1994

Telomerases.

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DNA-Directed DNA Polymerase↗
PubMed1993

Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes.

The telomere hypothesis of cellular aging proposes that loss of telomeric DNA (TTAGGG) from human chromosomes may ultimately cause cell-cycle exit during replicative senescence. Since lymphocytes have a limited replicative capacity and since blood cells were previously shown to lose telomeric DNA during aging in vivo, we wished to determine: (a) whether accelerated telomere loss is associated with the premature immunosenescence of lymphocytes in individuals with Down syndrome (DS) and (b) whether telomeric DNA is also lost during aging of lymphocytes in vitro. To investigate the effects of aging and trisomy 21 on telomere loss in vivo, genomic DNA was isolated from peripheral blood lymphocytes of 140 individuals (age 0-107 years), including 21 DS patients (age 0-45 years). Digestion with restriction enzymes HinfI and RsaI generated terminal restriction fragments (TRFs), which were detected by Southern analysis using a telomere-specific probe (32P-(C3TA2)3). The rate of telomere loss was calculated from the decrease in mean TRF length, as a function of donor age. DS patients showed a significantly higher rate of telomere loss with donor age (133 +/- 15 bp/year) compared with age-matched controls (41 +/- 7.7 bp/year) (P < .0005), suggesting that accelerated telomere loss is a biomarker of premature immunosenescence of DS patients and that it may play a role in this process. Telomere loss during aging in vitro was calculated for lymphocytes from four normal individuals, grown in culture for 10-30 population doublings. The rate of telomere loss was approximately 120 bp/cell doubling, comparable to that seen in other somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Telomere length predicts replicative capacity of human fibroblasts.

When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explained by the chronological age of the donor. Because we had previously shown that telomeres, the terminal guanine-rich sequences of chromosomes, shorten throughout the life-span of cultured cells, we wished to determine whether variation in initial telomere length would account for the unexplained variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93 yr) indicated relatively weak correlations between proliferative ability and donor age (m = -0.2 doubling per yr; r = -0.42; P = 0.02) and between telomeric DNA and donor age (m = -15 base pairs per yr; r = -0.43; P = 0.02). However, there was a striking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublings per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent significantly fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker of somatic cell aging in humans and are consistent with a causal role for telomere loss in this process. We also found that fibroblasts from Hutchinson-Gilford progeria donors had short telomeres, consistent with their reduced division potential in vitro. In contrast, telomeres from sperm DNA did not decrease with age of the donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.

Base Sequence↗
PubMed1992

Telomere end-replication problem and cell aging.

Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.

Adult↗
PubMed1992

Release of a transforming growth factor (TGF)-beta 2-related suppressor factor from postimplantation murine decidual tissue can be correlated with the detection of a subpopulation of cells containing RNA for TGF-beta 2.

Postimplantation murine decidual tissue from allopregnant C3H mice has been shown to release in vitro a potent immunosuppressive factor closely related to transforming growth factor (TGF)-beta 2 but slightly lower in apparent molecular weight. Decidual suppressor factor (DSF) activity was first detected in decidual tissue supernatant at day 9.5 of gestation and reached a plateau by day 10.5 to 12.5. By Northern analysis of decidual and placental tissue with a simian TGF-beta 2 probe, two characteristic TGF-beta 2 mRNA transcripts were detected in decidual tissue. In situ hybridization analysis of C3H implant sites, with the simian (pGEM-G1G2) TGF-beta 2 riboprobe, revealed a small population of TGF-beta 2+ cells localized to postimplantation decidua basalis and metrial gland cell area after day 8.5. On and before day 8.5, when DSF was not detectable, few TGF-beta 2 mRNA+ cells were detected. To test for TGF-beta release in situ, sections of uterine tissue were stained with antibody specific for TGF-beta 2, that identified DSF in Western blots. In postimplantation tissues (day 9.5, 12.5) patchy anti-TGF-beta 2 staining was seen over decidual tissue. Before day 9.5, slight and diffuse staining over decidual tissue was present with more marked staining of extradecidual tissue. Very little staining was noted over day 9.5 decidual tissue by using anti-TGF-beta 1 antibody as a control; however, some staining was seen over postimplantation fetal trophoblast and myometrial tissue. Fractionation of disaggregated postimplantation decidua by velocity sedimentation revealed that TGF-beta 2 mRNA+ cells were predominantly small and sedimented in the same fraction(s) as those cells previously shown to release DSF in vitro. Thus, the release of TGF-beta 2 related DSF correlates with the in situ detection of TGF-beta 2 mRNA and the in situ release of TGF-beta 2 peptide. These studies suggest that DSF may be a form of TGF-beta 2 released by a population of small lymphocytic decidual suppressor cells.

Animals↗
PubMed1992

Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.

Cell Transformation, Neoplastic↗
PubMed1992

Identification and characterization of a partial cDNA expressing interleukin-1-like activity in keratinocytes.

A partial cDNA has been isolated from the human keratinocyte cell line COLO-16 which is distinct from either IL-1 alpha or beta and encodes a protein which displays some of the biological properties associated with IL-1. The 720 bp partial cDNA hybridized on Northern blots of COLO-16 mRNA to a 1.6 kbp message of low abundance. Expression of the partial cDNA in COS-1 cells resulted in activity in three IL-1 assays: thymocyte co-stimulation, D10.G4.1 T-cell stimulation and fibroblast proliferation. Antisera generated against synthetic peptides based on inferred protein sequence from the cDNA reacted with a 20 kDa and 30 kDa species in both the COLO-16 cell line and PMA-stimulated normal human keratinocytes. These novel species were also present in PMA-stimulated and unstimulated human dermal fibroblasts and human T-cell lines.

Amino Acid Sequence↗
PubMed1992

Eosinophils in chronically inflamed human upper airway tissues express transforming growth factor beta 1 gene (TGF beta 1).

Transforming growth factor beta (TGF beta) is a multifunctional protein which has been suggested to play a central role in the pathogenesis of chronic inflammation and fibrosis. Nasal polyposis is a condition affecting the upper airways characterized by the presence of chronic inflammation and varying degrees of fibrosis. To examine the potential role of TGF beta in the pathogenesis of this condition, we investigated gene expression and cytokine production in nasal polyp tissues as well as in the normal nasal mucosa. By Northern blot analysis using a porcine TGF beta 1 cDNA probe, we detected TGF beta 1-specific mRNA in nasal polyp tissues, as well as in the tissue from a patient with allergic rhinitis, but not in the normal nasal mucosa. By the combination of tissue section staining with chromotrope 2R with in situ hybridization using the same TGF beta 1 probe, we found that approximately 50% of the eosinophils infiltrating the polyp tissue express the TGF beta 1 gene. In addition, immunohistochemical localization of TGF beta 1 was detected associated with extracellular matrix as well as in cells in the stroma. These results suggest that in nasal polyposis where eosinophils are the most prevalent inflammatory cell, TGF beta 1 synthesized by these cells may contribute to the structural abnormalities such as stromal fibrosis and basement membrane thickening which characterize this disease.

Chronic Disease↗