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