Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor Page: 3 of 16
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Senescence-Associated Secretory Phenotype
A
Growth Oxygen Fibroblast
st3tus % Birds
WI-38
MR-90
3% HCA-2
BJ
hBF
WI-38
IMR-90
20% HCA-2
BJ
hBF
WI-38
IMR-90
3% HCA-2
BJ
PRE hBF
[MR-RB
20% HCA-2
BJ
BEFB
- -
c7 rO < a O r? <aos - o-OC a uNumberof'R' Iy
- -N !N m m -112
1B
3
12
5
3
4
4i
W-3B (3% Oxygen)
0.5 10
2 4 7 10 2 4 7 10IL-6
IL-8 (CXCL-8)
GM-CSF
GRO-a (CXCL-1)
GRO-b (CXCL-2)
GRO-g (CXCL-3)
ICAM-1
Osteoprotegernn
MCP-1 (CCL-2)
MCP-2 (CCL-8)
MCP-3 (CCL-7)
+ - MCP-4 (CCL-13)
IGFBP-3 *
80% 5% p16
p21
p53o Fold change
w . relative to
o I PRE control
a NW. SEN SEN
I n (REP) (XRA)
65 68
898 495
393 492
84 195
32 30
145 74
13 18
11 3
23 20
27 38
10 30
16 17
11 10
5 2
6 5
2 2F
mRNA level
SASP factors soirel () = 064
non-SASP factors corral (r) = 0 15Figure 1. SASP of Human Fibroblasts
(A) Soluble factors secreted by the indicated cells were detected by antibody arrays and analyzed as described in the text, Materials and Methods; Text
S2; and Datasets S1-S4. For each cell strain, the PRE and SEN signals were averaged and used as the baseline. Senescence (SEN) was induced by either
repeatedly passaging the cells (REP, replicative exhaustion) or by exposing them to a relatively high dose (10 Gy) of ionizing radiation (XRA, X-
irradiation); for simplification, XRA and REP signals were averaged as a single SEN signal (see also Figure 1C). Signals higher than baseline are displayed
in yellow; signals below baseline are displayed in blue. The numbers on the heat map key (right) indicates log2-fold changes from the baseline.
(B) Validation by immunostaining. PRE WI-38 cells were made senescent by REP or XRA, maintained in 10% serum until fixation, and immunostained for
the cytokines IL-6 and IL-8 and the senescence marker p16INK4a.
(C) Correlation between the SASPs of WI-38 cells induced to senescence by XRA or REP, compared to PRE cells, depicted as log2-fold changes.
(D) WI-38 cells were X-irradiated at the indicated doses. CM was collected 2, 4, 7, or 10 d later, and soluble factors were analyzed by antibody arrays. PRE
cells and cells irradiated with 0.5 Gy transiently arrested growth, but resumed proliferation 24-48 h after CM collection. Cells irradiated with 10 Gy
became senescent, and therefore did proliferate during and beyond the course of the experiment. PRE and SEN (10 d) signals were averaged and used
as the baseline. Signals higher than baseline are displayed in yellow; signals below baseline are displayed in blue.
(E) PRE and SEN cells were analyzed for the indicated mRNAs by quantitative RT-PCR, and normalized to the corresponding PRE values (baselines). The
senescence inducers (REP or XRA) are given in parentheses. Signals higher than baselines are shown in red, signals below baselines are displayed in
green, and the fold changes in signals are given to the right of the heat map. The results are averages for four cell strains (WI-38, IMR90, HCA-2, and BJ),
and the total number of samples for each condition are given below the heat map. An asterisk (*) indicates non-SASP factors (see Figure S4).
(F) Correlation between mRNA and secreted protein levels for SASP (red) and non-SASP (blue) factors (see Figure S4). PRE and SEN values were averaged
to create a baseline; all values were expressed as the log2-fold change relative to this baseline; PRE versus baseline and SEN versus baseline are shown.
doi:10.1371/journal.pbio.0060301.g001(Datasets S1-S4) and for visual display (Figure IA). For the
visual display, values over baseline are displayed in grades of
yellow, and values under baseline are displayed in grades of
blue (Figure IA).
Although the display gives only a semiquantitative assess-
ment of how secretion levels vary (see accompanying scale in
Figure IA, with log2-fold changes indicated), the data (Data-
sets S1-S4) show that SEN cells secrete significantly higher
levels of numerous proteins compared to PRE cells (Figure
IA). We term this phenomenon a senescence-associated
secretory phenotype (SASP). Of 120 proteins interrogated
by the arrays, 41 were significantly altered in the SEN CM and
were oversecreted in comparison to PRE CM (Figure lA and
Dataset S4). However, the SASPs did not result from a general
PLoS Biology www.plosbiology.orgstimulation of secretion. Seventy-nine proteins showed no
significant differences in secreted levels between SEN and
PRE cells, although many of these proteins were easily
detectable by the arrays (Dataset S4).
The SASPs were complex, and their biological effects could
not be predicted a priori. SASP components included
inflammatory and immune-modulatory cytokines and chemo-
kines (e.g., IL-6, -7, and -8, MCP-2, and MIP-3a). They also
included growth factors (e.g., GRO, HGF, and IGFBPs), shed
cell surface molecules (e.g., ICAMs, uPAR, and TNF recep-
tors), and survival factors (Figure lA and Table S2). However,
the SASP was not a fixed phenotype. Rather, it was a wide-
ranging profile because each cell strain also displayed unique
quantitative or qualitative features. In addition, withinDecember 2008 Volume 6 Issue 12 e301
C
, -oD E
corral = 0 935
XRA dose (Gy)
Days after irradiation
IL-6
GRO (-a, -b, -g)
IL-8
GM-CSF
IL-7
GRO-a
ICAM-1
Osteopro tegerIn
HGF
IGFBP-4
Proliferation
BrdU labellingSEN (REP)
I
0
naB
80%N
.s
0003
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Coppé, Jean-Philippe; Patil, Christopher; Rodier, Francis; Sun, Yu; Munoz, Denise; Goldstein, Joshua et al. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor, article, October 24, 2008; Berkeley, California. (https://digital.library.unt.edu/ark:/67531/metadc902653/m1/3/: accessed March 29, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.