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Extension of Life-Span by Introduction of Telomerase Into Normal Human Cells

Extension of Life-Span by Introduction of Telomerase Into Normal Human Cells. By Micaile Brown Bodnar et al. 2000. Extension of life span by introduction of telomerase into normal human cells. Science: 279(5349):349. Introduction.

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Extension of Life-Span by Introduction of Telomerase Into Normal Human Cells

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  1. Extension of Life-Span by Introduction of Telomerase Into Normal Human Cells By Micaile Brown Bodnar et al. 2000. Extension of life span by introduction of telomerase into normal human cells. Science: 279(5349):349.

  2. Introduction • Varying research studies are being performed in order to understand the aging phenotype. • Aging is defined as the cumulative irreversible changes that take place in an organism over the life span following the attainment of maturity.

  3. Research has identified many processes thought to contribute to the aging phenotype. • This article will address one particular process thought to be associated with aging: Telomere Shortening.

  4. Background • Telomere: - repeated complex DNA sequences - located at the end of chromosomes - each specie shares a specific sequence - synthesized by telomerase (ribonucleoprotein enzyme)

  5. 2 Types of Telomere Sequences: • #1 Simple Telomeric Sequences: - located at the extreme ends of chromosomal DNA - consist of simple, relatively short, tandemly repeated sequences 2

  6. #2 Telomere Associated Sequences : - located at regions near, but not at the ends of chromosomes. - repeated, often complex DNA sequences 3

  7. Proposed Thought • Telomere loss is thought to control entry into senescence. • Senescence = Aging (Senescence is characterized as the stage when normal diploid cells enter a non-dividing state) • Senescence: - dependent upon cumulative cell divisions - Hence, a “mitotic clock” 4

  8. Cell Cycle

  9. Telomere Hypothesis of Cellular Aging • cells become senescent when progressive telomere shortening during each division produces a threshold telomere length. 5

  10. Purpose of Study • To determine if telomere shortening causes cellular senescence. 6

  11. Synthesis of Telomeric DNA by Telomerase

  12. Synthesis 2

  13. Synthesis 3 7

  14. Procedure: • Introduction of telomerase into normal human cells • Measurement of Telomere Lengthening • Lifespan Determination • ß-Galactosidase Staining

  15. Telomerase Introduction Into Normal Human Cells: • 2 telomerase negative human cell types were transfected with vectors encoding the human telomerase catalytic subunit (hTRT). • Cell types used: - Retinal pigment epithelial cells (RPE) - Foreskin fibroblast cells (BJ)

  16. Measurement of Telomere Lengthening • Measured Telomere lengths to determine if transfection of the hTRT had an effect on lengthening telomeres.

  17. Lifespan Determination • Determined if telomerase expression had an effect on cellular growth. • Hence, did the cells enter senescence or continue their cellular growth.

  18. ß-Galactosidase Staining • hTRT negative cells and hTRT positive cells were stained with senescence associated ß-galactosidase. • ß - galactosidase is a biomarker associated with cellular aging.

  19. Therefore, aging/senescent cell will show greater staining of ß-galactosidase than normal dividing cells.

  20. Results of Measurement of Telomere Lengthening • hTRT Negative Cells: showed a telomere length decrease of about .4 to 1.3kbp. • hTRT Positive Cells: showed a telomere increase of about 3.7kbp.

  21. Telomere Lengthening Results cont.: • Purpose: Determine if hTRT positive cells had greater telomere length than hTRT negative cells • Results: (RPE cells and Fibroblast cells) - hTRT + cells: had a greater mean terminal restriction fragment (TRF) length. - hTRT - cells: had a smaller TRF value

  22. TRF Length For hTRT+ and hTRT – Cells Retinal Pigment Epithelial Cells

  23. Results of Lifespan Determination • hTRT - Cells: * Slowed growth • hTRT + Cells: *Population of cell doubled about 20 times

  24. Results of ß-Galactosidase Staining • hTRT negative cells showed greater ß-Galactosidase staining than the hTRT positive cells. • Therefore, hTRT negative cells showed more senescence than the hTRT positive cells.

  25. Conclusions • Telomeres function in preventing chromosomal DNA from shortening. • The absence of telomerase catalytic subunit (hTRT) results in telomere shortening. • Telomere shortening is an intrinsic timing mechanism that controls the number of cell divisions prior to senescence.

  26. Hence, there seems to be a threshold level of telomerase activity that is required for life-span extension. • Without this level of telomerase activity, cells age/senesce.

  27. Combating Cellular Senescence • Can combat multiple aging conditions, such as: - wrinkling of the skin - atherosclerosis • Has important implications for biological research, the pharmaceutical industry, and medicine.

  28. References • Bodnar et al. 2000. Extension of life span by introduction of telomerase into normal human cells. Science: 279(5349):349. • Russell PJ. 2000. Fundamentals of Genetics. Addison Wesley Longman, Inc: p249.

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