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

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

  1. 1. Extension of Life-Span by Introduction of Telomerase into Normal Human Cells Andrea G. Bodnar et al. Science, pp. 349-352, vol. 279 (1998) Podlevsky, J.D., Bley, C.J., Omana, R.V., Qi, X., Chen, J. (2007) The Telomerase Database. Nucleic Acids Res. 36 D339-D343.
  2. 2. Telomeres senescence theory
  3. 3. Telomeres senescence theory Human telomeres (in yellow)
  4. 4. Telomeres senescence theory - TTAGGG/CCCTAA sequence - Synthesized by the ribonucleoprotein enzyme telomerase - Active in germline cells, keeps telomeres at about 15 kpb - Not expressed in most human Human telomeres (in yellow) somatic tissues, where telomeres lenghts is significantly shorter
  5. 5. Telomeres senescence theory - TTAGGG/CCCTAA sequence - Synthesized by the ribonucleoprotein enzyme telomerase - Active in germline cells, keeps telomeres at about 15 kpb - Not expressed in most human Human telomeres (in yellow) somatic tissues, where telomeres lenghts is significantly shorter Theory proposes that cells become senescent when progressive telomeres shortening during each division produces a treshold telomere length
  6. 6. How telomerase works
  7. 7. How telomerase works
  8. 8. How telomerase works
  9. 9. How telomerase works
  10. 10. How telomerase works
  11. 11. Research...
  12. 12. Research... - The hTRT has been cloned - telomerase activity can be reconstituted by transient expression of hTRT in norman human diploid cells - hTR (template RNA component costituvely expressed) - hTRT- normal cells transfected with 2 different hTRT expression constructs:
  13. 13. Research... - The hTRT has been cloned - telomerase activity can be reconstituted by transient expression of hTRT in norman human diploid cells - hTR (template RNA component costituvely expressed) - hTRT- normal cells transfected with 2 different hTRT expression constructs: #1 engineered by removal of the 5’ and 3’ untranslated regions of hTRT and creation of Kozak consensus sequence
  14. 14. Research... - The hTRT has been cloned - telomerase activity can be reconstituted by transient expression of hTRT in norman human diploid cells - hTR (template RNA component costituvely expressed) - hTRT- normal cells transfected with 2 different hTRT expression constructs: #1 engineered by removal of the 5’ and 3’ untranslated regions of hTRT and creation of Kozak consensus sequence #2 native sequence cloned downstream of the SV40 promoter
  15. 15. Research... - The hTRT has been cloned - telomerase activity can be reconstituted by transient expression of hTRT in norman human diploid cells - hTR (template RNA component costituvely expressed) - hTRT- normal cells transfected with 2 different hTRT expression constructs: #1 comparison between life span of MPSV-hTRT transfected cells and vector only transfected cells #2 comparison of lifespan of activity positive and activity negative stable clones containing SV40-hTRT constructs
  16. 16. Telomerase activity in stable Retinal Pigment Epithelial cells
  17. 17. Telomerase activity in stable Retinal Pigment Epithelial cells
  18. 18. Telomeres length in stable RPE and BJ clones
  19. 19. Telomeres length in stable RPE and BJ clones
  20. 20. Effects of telomerase expression on cell lifespan
  21. 21. Effects of telomerase expression on cell lifespan
  22. 22. Effects of telomerase expression on cell lifespan
  23. 23. Effects of telomerase expression on cell lifespan
  24. 24. Implications
  25. 25. Implications Results indicate that telomere loss in the absence of telomerase is the intrinsic timing mechanism that controls the number of cell divisions prior to senescence Very low level of telomerase activity are apparently insufficient to prevent telomere shortening. This is consistent with the observation that stem cells have low but detectable telomerase activity, yet continue to exhibit shortening of their telomeres throughout life Promoter strength, structure of untranslated regions, site of integration, levels of hTR and hTRT and telomere- or telomerase-associated proteins in specific cell types are all factors that may affect the functional level of telomerase
  26. 26. Ideas
  27. 27. Ideas - against atrophy of skin through loss of extracellular matrix homeostasis in dermal fibroblasts - age-related macular degeneration of lipofuscin and down- regulation of a neuronal survival factor in RPE cells - atherosclerosis caused by loss of proliferative capacity and overexpression of hypertensive and thrombotic factors in endothelial cells - replacing of tumor cell lines with cloned normal diploid cells - production of normal or engineered biotechnology products or gene therapy
  28. 28. Thank you for listening Original paper DOI: 10.1126/science.279.5349.349 pdf version available at http://www.slideshare.net/ATMB

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