Tetrapeptide Ala-Glu-Asp-Gly Induces Telomerase Activity and Telomere Elongation in Human Somatic Cells

V.K. Khavinson, I.E. Bondarev, A.A. Butyugov
Saint Petersburg Institute of Bioregulation and Gerontology, Northwest Branch of the Russian Academy of Medical Sciences

The addition of the tetrapeptide Ala-Gly-Asp-Gly to the culture of human fetal fibroblasts, negative for telomerase, induces the expression of the catalytic subunit, telomerase activity, and telomere elongation. This is likely associated with the reactivation of the telomerase gene in somatic cells, suggesting a potential increase in the lifespan of cellular populations and the organism as a whole.

Keywords: peptide, Ala-Glu-Asp-Gly, tetrapeptide, telomerase, telomeres, fibroblasts

Background

The tetrapeptide (Ala-Glu-Asp-Gly) was designed and synthesized based on the amino acid composition analysis of the peptide complex epitalon, extracted from the epiphysis of animal brains. It has been established that the tetrapeptide restores disrupted neuroendocrine regulation in elderly monkeys, induces ribosomal gene activation, and facilitates the decondensation of pericentromeric heterochromatin in lymphocytes from elderly humans, activating genes repressed due to age-related euchromatin condensation.

Epitalon has been shown to increase the median lifespan of mice and rats and reduce the frequency of chromosomal aberrations in SAM mice. Notably, the tetrapeptide increases the maximum lifespan without the development of malignant neoplasms in these animals. The telomerase theory of aging links the age-related decline in tissue proliferative potential to critical telomere shortening during cell division. Telomerase is a ribonucleoprotein enzyme that replenishes telomeric repeats lost due to DNA replication asymmetry, encoded by two genes for the RNA and protein components of the enzyme. In humans, telomerase expression and activity are observed in most malignant, germline, early embryonic, and possibly stem cells, but are absent in somatic cells.

This study investigated the effect of the tetrapeptide on the expression of the telomerase catalytic subunit, telomerase activity, and telomere elongation in human somatic cells.

Research Methodology

Cell cultures of the telomerase-positive line HeLa and primary culture of human fetal lung fibroblasts 602/17 (24 weeks) were obtained from the Cell Culture Laboratory of the Influenza Research Institute. Cells were cultured in DMEM medium with 10% fetal bovine serum, 2 mM L-glutamine, and 100 µg/mL gentamicin sulfate. Fetal fibroblasts, starting from the 27th passage, were treated with the tetrapeptide at a concentration of 0.05 µg/mL for 4 days and analyzed.

Immunohistochemistry:
To detect the expression of the telomerase catalytic subunit, fetal fibroblasts were placed on sterile slides and stained with mouse monoclonal antibodies to the human telomerase catalytic subunit. Secondary antibodies used were from a universal peroxidase ABC kit. Staining was conducted as per the reagent kit instructions, and microscopy was performed using a Nikon Eclipse E400 with an AST1 digital camera.

Telomerase Activity:
Telomerase activity was determined by telomere repeat amplification protocol (TRAP). Cells were washed, lysed, and the lysates centrifuged. Supernatants were used for telomerase activity analysis in vitro. PCR products were electrophoretically separated, stained with SYBR Green, and visualized under UV light.

Telomere Length Measurement:
The average telomere length in individual cells was measured using flow-FISH. Cells were hybridized with a FITC-labeled PNA probe specific for telomeres and analyzed by flow cytometry.

Statistical Analysis:
Data were processed using CellQuest and XnView1.17 software. Differences between groups were assessed using Student’s t-test.

Research Results

Immunohistochemistry revealed intense nuclear staining in telomerase-positive HeLa cells and fetal fibroblasts treated with the tetrapeptide. No such staining was observed in control fetal fibroblasts.

Telomerase activity was absent in control fetal fibroblasts but detected in HeLa cells and fibroblasts treated with the tetrapeptide.

Flow-FISH demonstrated an increase in the average maximum telomere length in G1 phase fetal fibroblasts treated with Epitalon compared to control cells. The average telomere length in the control was 180 units, which increased to 240 units with tetrapeptide treatment (p<0.05).

The experiment demonstrated that the tetrapeptide can induce telomerase catalytic subunit expression, telomerase activity, and telomere elongation in human somatic cells, with an average increase of 33.3%. This is the first demonstration of telomerase activity induction by a peptide, contributing to the understanding of the geroprotective effects of the tetrapeptide in various experimental models. Critically short telomeres cannot prevent chromosomal fusion, which may activate proto-oncogenes and lead to cell malignancy.

The activation of telomerase and telomere elongation by the peptide explains its antitumor effect in aged animals.

References:

1. Mikhailov S.V., Boher K.P., Khavinson V.K., Anisimov V.N. // Bull. Exp. Biol. 2002. Vol. 133, No. 3. pp. 340-347.
2. Rosenfeld S.V., Togo E.F., Mikheev V.S., et al. // Ibid. pp. 320-322.
3. Khavinson V.K., Lezhava T-A., Monaselidze D.G., et al. // Ibid. No. 10. pp. 451-455.
4. Anisimov V.N., Khavinson V.K., Mikhailov A.I., et al. // Mech. Aging Dev. 2001. Vol. 122. pp. 41-68.
5. Anisimov V.N., Khavinson V.K., Popovich I.G., Zabezhinski M.A. // Cancer Lett. 2002. Vol. 183. pp. 1-8.
6. Anisimov V.N., Khavinson V.K., Provinciali M., et al. // Int. J. Cancer. 2002. Vol. 101. pp. 7-10.
7. Elmore L.W., Holt S.E. // Mol. Carcinog. 2000. Vol. 28, No. 1. pp. 1-4.
8. Peng J., Funk W.D., Wang S.S., et al. // Science. 1995. Vol. 269, No. 5228. pp. 1236-1241.
9. Hiyama E., Hiyama K., Yokoyama T., et al. // Nat. Med. 1995. Vol. 1. pp. 249-255.
10. Khavinson V.K. // Neuroendocrinol. Lett. 2002. Vol. 23, Suppl. 3. pp. 11-144.
11. Khavinson V., Goncharova N.D., Lapin B. // Ibid. 2001. Vol. 22. pp. 251-254.
12. Meyerson M., Counter C., Eaton E.N. // Cell. 1997. Vol. 90, No. 4. pp. 785-795.
13. Morin G.B. // Cell. 1989. Vol. 59, No. 3. pp. 521-529.
14. Rufer N., Dragowska W., Thompson G., et al. // Nat. Biotechnol. 1998. Vol. 16, No. 8. pp. 743-747.
15. Wright W.E., Shay J.W. // Nat. Biotechnol. 2002. Vol. 20, No. 7. pp. 682-688.

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