Extension of LifeSpan by Introduction of Telomerase Into Normal Human Cells

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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.

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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.

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• 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.

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Background

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

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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

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

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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

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Cell Cycle
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Telomere Hypothesis of Cellular Aging

• cells become senescent when progressive telomere
  shortening during each division produces a
  threshold telomere length.

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Purpose of Study

• To determine if telomere shortening causes
  cellular senescence.

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Synthesis of Telomeric DNA by Telomerase
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Synthesis 2
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Synthesis 3
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Procedure

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

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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)

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Measurement of Telomere Lengthening

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

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Lifespan Determination

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

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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.

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

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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.

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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

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TRF Length For hTRT and hTRT Cells
Retinal Pigment Epithelial Cells
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(No Transcript)
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Results of Lifespan Determination

• hTRT - Cells
• Slowed growth
• hTRT Cells
• Population of cell doubled
  about
• 20 times

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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.

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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.

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• 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.

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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.

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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.