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There is substantial evidence that aging is related to the finite ability of somatic cells to divide and repair damaged tissue. Since the seminal observation of Hayflick and Moor head (1) that cultured human fibroblasts have a finite replicative lifespan, a great deal of basic biological research on aging has been based on the model of cellular senescence in vitro (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.

Telomere dynamics and changes in telomerase activity are consistent elements of cellular alterations associated with changes in proliferative state. In particular, the highly specific correlations and early causal relationships between telomere loss in the absence of telomerase activity and replicative senescence or crisis, on the one hand, and telomerase reactivation and cell immortality, on the other, point to a new and important paradigm in the complementary fields of ageing and cancer. Although the signalling pathways between telomeres and transcriptional and cell cycle machinery remain undefined, recently described homologies between telomeric proteins and lipid/protein kinase activities important in chromosome stability provide evidence for the existence of pathways transducing signals originating in chromosome structure to cell cycle regulatory processes. Similarities between cell cycle arrest at senescence and the response of mortal cells to DNA/oxidative damage suggest overlap in the signal transduction mechanisms culminating in irreversible and stable cell cycle arrest. The feasibility of targeting telomeres/telomerase as a strategy for antiproliferative therapeutics has been shown in studies in yeast, in which mutations in specific telomere associated genes result in delayed cell death. Similarly, antisense oligonucleotide inhibition of telomerase activity in human tumour cells (HeLa) results in delayed cell death. The mechanism of cell death and possible escape from this fate require further study. In human cells, however, it would seem reasonable to predict that in these circumstances, apoptosis is induced in the vast majority of cells either directly in response to a DNA damage signal arising from critically shortened telomeres or as a secondary consequence of genetic instability.

Telomerase expression is repressed early in development in all normal somatic human tissues investigated to date, whereas activity and the expression of the RNA component for this enzyme are upregulated in almost all cases of malignant transformation and late-stage cancer. The telomere hypothesis of ageing and immortalization postulates that sufficient telomere loss on one or more chromosomes in normal somatic cells triggers cell senescence, whereas reactivation of the enzyme is necessary for cell immortalization. Measurements of telomere length and telomerase activity in cancer and during normal and accelerated human ageing in skin, blood, haemopoietic, skeletal muscle, vascular and CNS tissues support this model. Tissue culture studies of cell ageing and transformation have added to our understanding of telomere dynamics in these processes. Evolution of telomerase repression and mortality in somatic cells of long-lived organisms is consistent with antagonistic pleiotropy models in which cell senescence is a tumour suppressor mechanism: stringent repression of telomerase has a beneficial early effect in reducing the probability of cancer, but a deleterious, unselected late effect in its contributions to age-related disease.

The data reviewed here suggest that telomere dynamics and telomerase expression are fundamentally involved in cellular aging and cancer. Of particular importance is the stabilization of telomeres by activation of telomerase and the association of this process with cell immortality and human malignancies. Thus, we believe that cell immortalization is required for long-term growth of the vast majority of malignant or metastatic tumors and that advances in telomere biology and telomerase inhibition will improve the way cancers are diagnosed and treated. We look forward to the clinical evaluation of these bold predictions.

Telomere shortening may contribute to the limited lifespan of somatic cells and telomerase, the enzyme that elongates telomeric DNA and maintains telomere length, may be essential for unlimited cell proliferation in vivo and in vitro. Telomerase is not expressed in most human somatic cells but is a nearly ubiquitous tumour marker, being activated in malignant cells from many cancers. Inhibition of telomerase may lead to telomere shortening and eventually limit the proliferative capacity of malignant cells and hence be of therapeutic value. With the intent of characterizing an animal model for inhibition studies, we investigated telomerase activity during mammary tumorigenesis in transgenic mice overexpressing the neu gene. We detected activity in primary mammary tumours and lung metastases but also in normal mammary glands and other organs. Activity was elevated in tumors versus normal tissues and was enhanced by short-term culturing of normal cells. Telomerase activity was also present in somatic tissues from the non-transgenic parental strain and the outbred Mus spretus strain. As we recently detected telomerase activity in normal human hemopoietic tissues, mouse models of tumorigenesis may provide useful experimental systems for assessing the outcome of in vivo inhibition of telomerase in both malignant and normal cells.

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)

The human keratinocyte cell line COLO-16 expresses mRNA homologous to human IL-1 alpha and IL-beta (transcript sizes 2.3 and 1.6 kb, respectively). A 1.2 kbp cDNA was selected with a human IL-1 beta probe from a lambda gt11 library constructed using poly A+ RNA from COLO-16 cells. Sequence analysis revealed that this cDNA was nearly identical to the 3' 1.2 kb of human monocyte IL-1 beta. When this cDNA was expressed in COS cells using a mammalian expression vector, IL-1 activity was detected in the cell conditioned supernatants using assays for D10-T-cell, thymocyte and fibroblast proliferation. Western analysis of lysates from COS cells transfected with this clone revealed the presence of a -17 kDa protein which reacted with antisera to human IL-1 beta. This protein was the same size as the processed form of IL-1 beta present in COLO-16 cells suggesting that this cDNA encodes the mature form of IL-1 beta. COLO-16 cells contain proteins of -30 kDa and 17 kDa which are immunoreactive with specific antisera for human IL-1 alpha and human IL-1 beta. Despite the presence of four-fold greater amounts of immunoreactive IL-1 beta protein than IL-1 alpha in cell lysates, all the IL-1 activity in the lysate could be neutralized by antisera to IL-1 alpha. IL-1 beta comprised only 25% of the IL-1 activity in the cell-conditioned media, all remaining activity was neutralized by antisera to IL-1 alpha. Whereas IL-1 alpha protein in both cell lysates and conditioned supernatants was predominantly in the processed -17 kDa form, IL-1 beta proteins were primarily of the processed and inactive 30kDa species. This apparent inability of keratinocytes to process IL-1 beta may explain our observations that the IL-1 activity secreted by COLO-16 cells is principally due to IL-1 alpha.

3168 Background: Telomerase maintains telomeric DNA and plays a critical role in tumor cell immortality. Human telomerase is repressed or transiently active in normal somatic cells and telomeres gr...

Acute starvation of mammalian cells for amino acids results in translational errors that may be detected by two-dimensional polyacrylamide gel electrophoresis. Using this as an assay for error frequency in mammalian cells, we investigated the hypothesis that neoplastic transformation was associated with an increased error frequency which in turn leads to an increased mutation rate and a decreased efficiency of regulatory controls (phenomena of tumor progression). Although we found that transformation was not always associated with an increased level of mistranslation we showed that SV40 transformation increased the level of translational errors in all cell types tested.

The complete cDNA for a human mitochondrial protein designated P1, which was previously identified as a microtubule-related protein, has been cloned and sequenced. The deduced amino acid sequence of P1 shows strong homology (40 to 50% identical residues and an additional 20% conservative replacements) to the 65-kilodalton major antigen of mycobacteria, to the GroEL protein of Escherichia coli, and to the ribulose 1,5-bisphosphate carboxylase-oxygenase (rubisco) subunit binding protein of plant chloroplasts. Similar to the case with the latter two proteins, which have been shown to act as chaperonins in the posttranslational assembly of oligomeric protein structures, it is suggested that P1 may play a similar role in mammalian cells. The observed high degree of homology between human P1 and mycobacterial antigen also suggests the possible involvement of this protein in certain autoimmune diseases.

The fidelity of protein synthesis was measured in human diploid skin fibroblasts as a function of passage level ("aging in vitro") and physiological age of tissue donor ("aging in vivo") using two different test systems. First, in cell-free extracts the ratio of delta leu/delta phe incorporation into peptide linkage following in the latter case using cells derived from elderly normal donors and from subjects with the premature aging disorders of Hutchinson-Gilford progeria and the Werner syndrome. Similar results were obtained using a second system of intact cells whereby histidine starvation induces quantifiable satellite spots resolved by two dimensional electrophoresis on polyacrylamide gels on the acidic side of the native actin species due to substitution of the neutral amino acid glutamine for the basic histidine. In fact, error frequencies appeared to decrease during aging in vitro, likely due to selection for clonal subpopulations with the highest fidelity of protein synthesis. The only increases were seen in the intact cell system where SV40-transformed cells showed a three-to-five fold greater error frequency compared to nontransformed fibroblasts. In total, these data fail to support the error catastrophe theory of cellular aging.