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- –1 st - cellular senescence may be an anti-cancer mechanism or tumor suppressive mechanism (+) –2 nd - 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. SA B-gal B-galactosidase staining at pH 6 on normal WI38 cells at population doubling 29 (left) and senescent WI38 cells at population doubling 36 (right).

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