development and validation of an in vitro model for osteoblast aging: beta- galactosidase and...
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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
• 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.
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
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.
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
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)
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
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.
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)
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).
Relationship of Beta-galactosidase/ Acridine Orange and Various Conditions
Expected Beta-gal positivity for given condition
0
20
40
60
80
100
120
Young, LogGrowth
Young, Quiescent Toxin Exposed Senescent Cells
Cell state
Bet
a-g
al +
Series1
Expected relationship of AO + stained nucleoli and given condition
0
20
40
60
80
100
120
Young, LogGrowth
Young, Quiescent Toxin Exposed Senescent Cells
Cell state
0-2 nucleoli
≥3 nucleoli
• 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
Results- Skin Fibroblasts (AG11016)
X gal and Acridine Orange in Quiescent AG11016 C26.65 cells
10.00%
90.00%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
0-2 ≥3
# nucleoli
% X
gal
+ a
nd
AO
+
X-gal + and Acridine Orange staining in AG11016 C24.16 exposed to 50uM H2O2 for 1 week
7%
93%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0-2 ≥3
# of nucleoli
% X
-gal
+/A
crid
ine
Ora
ng
e
AO in AG11016 C10.13 human skin fibriblasts in vitro
3.50%
96.50%
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
120.00%
0-2 ≥3
# of nucleoli
Beta-gal + and AO + in AG11016 C10.13 human skin fobroblasts in vitro (N=5)
0%
100%
0%
20%
40%
60%
80%
100%
120%
0-2 ≥3
# of nucleoli
% b-ga
l + and
AO+
Beta-gal + and AO + in AG11016 C
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0-2 ≥3
# nucleoli
% b
-gal
+ a
nd
AO
+
Results- AG11016
Results- Osteoblasts (NHOST)X gal and Acridone Orange in Quiescent NHOST C25.95 cells
15.00%
85.00%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
0-2 ≥3
# nucleoli
% X
gal
+ a
nd
AO
+
15
Xgal + and AO in NHOST C25.95 exposed to 50uM H2O2 x 1 week
26.50%
73.50%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
0-2 ≥3
# of nucleoli
% X
gal
+ a
nd
AO
Beta-gal + and AO + in NHOST C
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0-2 ≥3
# nucleoli
% b
-gal
+ a
nd
AO
+
Beta-gal + and AO + in NHOST C
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0-2 ≥3
# nucleoli
% b
-gal
+ a
nd
AO
+
Results- NHOST
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