TRANSIENT TELOMERASE EXPRESSION IN NORMAL SOMATIC CELLS LEADS TO TELOMERE EXTENSION AND INCREASED PROLIFERATION IN THE ABSENCE OF MALIGNANT TRANSFORMATION
Bibliographic record
Abstract
O276* Aims: Progressive loss of telomeres through cell division leads to cell cycle arrest and apoptosis rendering cells incapable of unlimited tissue repair. This limited capacity reduces allograft survival as a result of injury from acute and chronic rejection. In this study, normal endothelial cells were rescued from telomere shortening via the transient expression of human telomerase (hTERT) to reverse replicative senescence. We hypothesized that this transient telomerase activity imparts a growth advantage to these cells via telomere maintenance without causing a malignant phenotype. Methods: Telomerase activity in hTERT-transfected normal human umbilical vein endothelial cells (HUVECs) was assessed using the telomeric repeat amplification protocol (TRAP). Telomere length in HUVECs was examined before and after telomerase transfection with Effectene. A flow-FISH procedure was used with a FITC-labelled telomere-specific PNA probe containing sequence complementary to telomeric DNA. The signal intensity of fluorescence was standardized into molecules of equivalent soluble fluorochrome (MESF) unit (M) that directly correlates to telomere length. HeLa cells was used as controls in the different assays that evaluate telomerase activity, as well as biological function and malignant transformation utilizing standard protocols in soft agar and matrigel respectively. Results: A decline in telomere length, as a function of culture passage, was detected in normal aging HUVECs. HUVECs, at escalating passage numbers (p), showed mean MESF values of 53±16.0 kM (n=4):p5-7, 40±7.8 kM (n=6):p10-13, 28±5.3 kM (n=5):p14-17, 24±3.5 kM (n=4):p18-20 and 20±3.2 kM (n=3):p21-23. Following hTERT transfection, variable telomere extension was found in both young and senescing cultures whose DNA uptake efficiency was not significantly hampered by age (17±2.52% vs 12±1.55%, p>0.05). The net increase in telomere length ranged from 2 to 222 kM for HUVECs between p5 and p23. Telomerase activity peaked on day 2 post-transfection and returned to baseline levels, similar to untransfected and heat-denatured controls, on day 5. Peaked activity was 4% of that observed in HeLa positive controls. Whereas HeLa cells lacked tubule formation, hTERT-expressing HUVECs demonstrated vessel sprouting on matrigel, which was also observed with normal HUVECs. Only HeLa cells formed colonies on soft agar indicative of malignant transformation. Although untransformed, hTERT-transfected HUVECs showed an increase in proliferation. Proliferation index in terms of %S-phase cycling cells in hTERT-transfected cultures displayed a net increase of up to 7% despite in-vitro aging. Such an increase led to a more rapid cell recovery following serum starvation compared to HUVECs transfected with an irrelevant β-galactosidase vector. Conclusions: Transient telomerase expression leads to telomere lengthening in normal somatic cells, which can potentially increase cellular lifespan. While telomerase activity boost cell proliferation, it does not cause malignant transformation rendering it a feasible gene therapeutic strategy to prolong allograft survival.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".