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Andrew Rosenzweig, MD Lab Meeting 2.26.08

Development and Validation of an In Vitro Model for Osteoblast Aging: Beta-galactosidase and Acridine Orange. Andrew Rosenzweig, MD Lab Meeting 2.26.08. Goals.

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Andrew Rosenzweig, MD Lab Meeting 2.26.08

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  1. Development and Validation of an In Vitro Model for Osteoblast Aging: Beta-galactosidase and Acridine Orange Andrew Rosenzweig, MD Lab Meeting 2.26.08

  2. Goals • Develop a methodology to identify truly senescent cells in vitro (and eventually in vivo)with higher sensitivity than previously reported assays. • Use this protocol to justify the theory that cellular aging occurs in vivo and contributes to age-related pathologic processes.

  3. Background- Cellular Senescence • Hayflick 1961- Normal, somatic cells do not divide indefinitely but have a finite replicative lifespan. • Senescent cells are characterized by an inability to progress through the cell cycle, usually with a DNA content consistent with late G1. Cells remain metabolically active but fail to initiate DNA initiation. • In contrast to quiescence (G0) where growth arrest is not permanent and cells may resume proliferation in response to appropriate signals • Irreversibly growth-arrested cells survive for long periods of time without any obvious signs of cell death (apoptosis resistance). • At the molecular level, the tumor suppressor genes pRb/p16 and p53/p21 control cellular senescence

  4. Background • Two seemingly competing hypotheses- • 1st- cellular senescence may be an anti-cancer mechanism or tumor suppressive mechanism (+) • 2nd- tissue regeneration and repair deteriorate with age; senescence may promote aging (-) • Antagonistic Pleiotropy- genes or processes that were selected to benefit the health and fitness of young organisms can have unselected deleterious effects that manifest in older organisms and thereby contribute to aging • Relationship between cellular senescence in culture and aging in vivo is still not clear.

  5. Senescence markers • Several markers can identify senescent cells in culture and in vivo but none are exclusive to the senescent state • (lack of) DNA replication- i.e. BrdU, 3H-Thymidine, • senescence associated B-galactosidase- induced by stressors ie toxins, confluence • Probably reflects increase in lysosomal biogenesis commonly occurring in senescence • p16- expressed by many but not all senescent cells and also expressed by some tumor cells

  6. Senescence markers • Senescence associated heterchromatin foci (SAHFs)- chromatin structure is reorganized leading to transcriptional silencing of growth-promoting genes • Preferential binding of DNA dyes- i.e. DAPI, HP1, Acridine Orange • Senescence associated DNA damage foci- dysfunctional telomeres and other sources pf DNA damageγH2AX and 53BP1 • New markers related to oncogene-induced senescence- differentiated embryo-chondrocyte expressed-1 (DEC1), p15 (a CDK1) and decoy death receptor-2 (DCR2)

  7. Hypothesis • Using markers for senescence in culture and in human bone samples, we hypothesize that these models will allow further insight into osteoblast aging in vivo

  8. Acridine Orange • The proposed arrest of senescent cells in late G1/S can be observed by chromatin condensation patterns • Tips of 5 pairs of chromosomes (up to 10 fragments after last mitosis) fuse into fewer and larger fragments as they approach S phase • As cells progress through the cell cycle the fraction of cells containing 1 or 2 nucleolar fragments while the fraction containing 3 or more fragments decreases • Up to 90% of senescent cells in culture may contain only 1 to 2 nucleolar fragments • Acridine orange- binds to nucleoli and allows them to be counted.

  9. Senescence Associated β-Galactosidase (SA B-gal) • B-galactosidase is a eukaryotic hydrolase enzyme localized in the lysosome that catalyzes the hydrolysis of B-galactosides to monosaccharides • A B-galactosidase-related protein with no detectable enzymatic activity has been described in a variety of human tissues • Origin and function still unknown • Potential marker for senescence of fibroblast cultures in vitro • SA B-gal at pH 6.0 has been reported to increase during replicative senescence and may reflect replicative/physiologic age of cells (although not necessarily the chronologic age of the donor)- Dimri et al. 1995 • Limited application b/c not specific to senescence- • Also increased in quiescent, immortalized and serum starved cells • Reversible under other conditions • May actually be lysosomal enzyme releases at suboptimal pH (4.0)

  10. B-galactosidase staining at pH 6 on normal WI38 cells at population doubling 29 (left) and senescent WI38 cells at population doubling 36 (right). SA B-gal

  11. Relationship of Beta-galactosidase/ Acridine Orange and Various Conditions • Young, log growing cells- little/no B-gal; ≥ 3 nucleoli (AO) • Quiescent cells- incubated with 0.01%FBS x 4 days- little/no B-gal; ≥3 nucluoli • Toxin-exposed cells- xx H2O2 x 7 days- little/no B-gal; ≥3 nucleoli • Senescent cells- +B-gal; 0-2 nucleoli

  12. Results- Skin Fibroblasts (AG11016)

  13. Results- AG11016

  14. Results- Osteoblasts (NHOST)

  15. Results- NHOST

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