We thank Hernandez Borrero and colleagues for their comments (1) on our article (2), especially their consideration of novel paradigm-shifting aspects. As they requested, we have presented coimmunoprecipitation data showing endogenous p53–p21 protein interaction in IMR-32 and H460 cells (Fig. 1). Levels of the p53–p21 complex elevated upon γ-irradiation of H460 cells, consistent with the ability of γ-irradiation to increase p53 and p21 levels. We have additionally investigated whether p21 influences cell growth under our experimental conditions. We performed cell invasion assays by seeding cells in serum-free medium onto the upper surface of Matrigel-coated filters in Transwell chambers and filling the lower chambers with medium containing FBS (10%). After approximately 16 hours of incubation, we counted the cells that migrated to the lower surface of filters. Considering diffusion of FBS through the filters, cells were exposed to 0% to 10% FBS. We found that p21 knockdown with or without cytoplasmic p53 (p53K305N) did not significantly influence cell growth in serum-free and 10% FBS-containing media (Fig. 2), suggesting that p21 can regulate cell invasion without significantly altering cell growth. Alterations in the levels of p53 or p21 expression did not significantly influence cell growth during similar incubation periods (3).

Figure 1.

Interactions of endogenous proteins p53 and p21 in IMR-32 neuroblastoma and H460 lung cancer cells. A, IMR-32 cell lysates were immunoprecipitated (IP) using an anti-p21 antibody. Level of expressed p53 in the precipitates and input controls were compared by Western blotting (WB) using β-actin as a loading control. In the first lane, IgG serves as a negative control of immunoprecipitation. B, The same experiments were repeated using lysates of untreated control and γ-irradiated (10 Gy, 3-hour postirradiation) H460 cells.

Figure 1.

Interactions of endogenous proteins p53 and p21 in IMR-32 neuroblastoma and H460 lung cancer cells. A, IMR-32 cell lysates were immunoprecipitated (IP) using an anti-p21 antibody. Level of expressed p53 in the precipitates and input controls were compared by Western blotting (WB) using β-actin as a loading control. In the first lane, IgG serves as a negative control of immunoprecipitation. B, The same experiments were repeated using lysates of untreated control and γ-irradiated (10 Gy, 3-hour postirradiation) H460 cells.

Close modal
Figure 2.

p21 and p53K305N do not significantly influence cell growth. A and B, Empty or p53K305N-encoding pcDNA3 vectors and control and p21-targeting siRNAs were introduced into H1299 cells in the indicated combinations. After 24-hour recovery, an equal number of cells (5 × 104) was seeded in culture plates with a FBS (10%)-containing or serum-free medium. The number of viable cells was compared by trypan blue staining (A) or by MTT assay (B) after 16- and 24-hour incubation.

Figure 2.

p21 and p53K305N do not significantly influence cell growth. A and B, Empty or p53K305N-encoding pcDNA3 vectors and control and p21-targeting siRNAs were introduced into H1299 cells in the indicated combinations. After 24-hour recovery, an equal number of cells (5 × 104) was seeded in culture plates with a FBS (10%)-containing or serum-free medium. The number of viable cells was compared by trypan blue staining (A) or by MTT assay (B) after 16- and 24-hour incubation.

Close modal

A major finding of our article is that the p53/p21 complex is a functional unit that can act on multiple cellular components and that prosurvival Bcl-2 proteins are targets of the p53/p21 complex. p53 and p21 may form complexes with many other proteins. We discussed the possibility that the p53/p21 complex regulates cell functions other than invasion and apoptosis by binding to other targets. The binding of the p53/p21 complex to its targets may depend on various factors, including subcellular localization of p53 and p21, cellular physiology, and extracellular stressors.

Bax can exert nonapoptotic activity, apart from its proapoptotic activity (4). We demonstrated that knockdown or knockout of Bax promotes cell invasion (5) and that Bax induced by p53 overexpression consistently suppresses cell invasion without significantly altering cellular viability (6). This nuclear function of p53 is distinguished from the function of cytoplasmic p53, which suppresses cell invasion by liberating Bax from prosurvival Bcl-2 proteins (6). Previously, we demonstrated that the p53/p21 complex promotes Mdm2-dependent Slug ubiquitination and degradation (7).

Regarding the criticism that p53 levels in Fig. 5B of the original article (2) are inconsistent with the paradigm in the p53 field, we present time-course levels of total p53 in p53-H1299 transfectants, showing that p53 levels increased at 3 to 12 hours, but returned nearly to the original level at 24 hours after irradiation (Fig. 3). We believe that this is a general pattern of p53 stabilization and downregulation in response to DNA-damaging agents. Moreover, p53 levels can respond to DNA-damaging agents in different dynamics depending on its subcellular localization (cytosol or nucleus; ref. 8). Furthermore, decrease and increase in nuclear and cytosolic p53 levels, respectively, upon apoptotic stimulation, has been reported by other investigators (9). Hernandez Borrero and colleagues additionally pointed out that despite the cell growth–suppressing activity of p53, p53-expressing and -null tumors showed similar growth in mice (Fig. 6A; ref. 2). This experiment was performed by selecting xenograft tumors of p53-null and p53-expressing H1299 cells at a similar size (∼200 mm3), as we described in the Materials and Methods of ref. 2. Therefore, p53 levels in the selected p53-H1299 tumors might be too low to significantly influence tumor growth, unless they were irradiated. We repeated the experiments using p53-null and p53-expressing HCT116 colon cancer cells and obtained similar results (Supplementary Fig. S5; ref. 2). Our results are consistent with the report that p53-null and p53-expressing HCT116 tumors grew at similar rates in mice unless they were exposed to genotoxic drugs (10) or metabolic stresses (11). The apoptotic effects of p53 were not mimicked by p53ΔC37. Nevertheless, similar to p53, p53ΔC37 is localized mainly in the nucleus and relocates to the cytosol after γ-irradiation (Fig. 5E; ref. 2). These data indicate that cytosolic p53 requires its interaction with p21 to induce cell death, as further supported by the finding that unlike p53, p53ΔC37 failed to dissociate the Bcl-w/Bax complex, an event occurring outside the nucleus (Fig. 5E; ref. 2).

Figure 3.

Responses of total p53 levels in p53-H1299 transfectants to γ-irradiation. H1299 cells were transfected with empty or p53-encoding pcDNA vector. The p53-H1299 transfectants were γ-irradiated (20 Gy). At the indicated postirradiation times, levels of p53 in cell lysates were compared by Western blotting.

Figure 3.

Responses of total p53 levels in p53-H1299 transfectants to γ-irradiation. H1299 cells were transfected with empty or p53-encoding pcDNA vector. The p53-H1299 transfectants were γ-irradiated (20 Gy). At the indicated postirradiation times, levels of p53 in cell lysates were compared by Western blotting.

Close modal

Hernandez Borrero and colleagues presented the data that p21 ablation does not increase the migration of SW780 bladder cancer cells (1), in contrast to our model (Fig. 7; ref. 2). We stated that the model may represent a general phenomenon, as supported by our data from diverse cancer cell types, including lung, colon, and neuroblastoma cells. This does not necessarily mean that the model applies to all cancer cells, which are highly diverse. The reason why p21 ablation did not increase SW780 cell migration is not clear. Damage to cells or alterations in the downstream pathways of the p53/p21 complex are possibilities.

p21 can inhibit or stimulate cell death, although the determinants of these opposing functions are unclear yet. Originally (2), we demonstrated proapoptotic function of p21 using two types of apoptotic stimuli (γ-irradiation and H2O2) and multiple types of cancer cells (H1299, Calu-1, and HCT116). The proapoptotic function of p21 in γ-irradiation–induced death of mouse embryonic fibroblasts (12) and thymocytes (13) has been reported. Notably, in the latter case, p21 lost its proapoptotic function after p53 knockout, thus supporting participation of the p53/p21 complex in γ-irradiation–induced apoptosis. Moreover, both p53 and p21 are required for drug-induced colon cancer cell death (14). These reports support our opinion that proapoptotic function of the p53/p21 complex is not limited to a specific experimental setting.

In conclusion, we would like to clarify that the p53/p21 complex is a functional unit that acts on multiple cell components, and its action on Bcl-2 proteins contributes to the ability of p53 and p21 to suppress cell invasion and promote cell death. Identification of new targets of the p53/p21 complex and analysis of the relationship among these targets require further research.

See the original Letter to the Editor, p. 2770

No potential conflicts of interest were disclosed.

This work was supported by grants from the National Research Foundation of Korea (2017R1D1A1B03032395) and the Korea Institute of Radiological and Medical Sciences, funded by the Ministry of Sciences and ICT, Republic of Korea (50531-2018).

1.
Hernandez Borrero
LJ
,
Sikder
R
,
Lulla
A
,
Gokare
P
,
Del Valle
PR
,
Tian
X
, et al
Bcl-2 protein targeting by the p53/p21 complex—letter.
Cancer Res
2018
;
78
:
2770
1
.
2.
Kim
EM
,
Jung
CH
,
Kim
J
,
Hwang
SG
,
Park
JK
,
Um
HD
. 
The p53/p21 complex regulates cancer cell invasion and apoptosis by targeting Bcl-2 family proteins
.
Cancer Res
2017
;
77
:
3092
100
.
3.
Zemskova
M
,
Lilly
MB
,
Lin
YW
,
Song
JH
,
Kraft
AS
. 
p53-dependent induction of prostate cancer cell senescence by the PIM1 protein kinase
.
Mol Cancer Res
2010
;
8
:
1126
41
.
4.
Boohaker
RJ
,
Zhang
G
,
Carlson
AL
,
Nemec
KN
,
Khaled
AR
. 
Bax supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells
.
Am J Physiol Cell Physiol
2011
;
300
:
C1466
78
.
5.
Kim
EM
,
Kim
J
,
Park
JK
,
Hwang
SG
,
Kim
WJ
,
Lee
WJ
, et al
Bcl-w promotes cell invasion by blocking the invasion-suppressing action of Bax
.
Cell Signal
2012
;
24
:
1163
72
.
6.
Kim
EM
,
Park
JK
,
Hwang
SG
,
Kim
WJ
,
Liu
ZG
,
Kang
SW
, et al
Nuclear and cytoplasmic p53 suppress cell invasion by inhibiting respiratory complex-I activity via Bcl-2 family proteins
.
Oncotarget
2014
;
5
:
8452
65
.
7.
Kim
J
,
Bae
S
,
An
S
,
Park
JK
,
Kim
EM
,
Hwang
SG
, et al
Cooperative actions of p21WAF1 and p53 induce Slug protein degradation and suppress cell invasion
.
EMBO Rep
2014
;
15
:
1062
8
.
8.
Abdullah
A
,
Sane
S
,
Freeling
JL
,
Wang
H
,
Zhang
D
,
Rezvani
K
. 
Nucleocytoplasmic translocation of UBXN2A is required for apoptosis during DNA damage stresses in colon cancer cells
.
J Cancer
2015
;
6
:
1066
78
,
9.
Heo
KS
,
Lee
H
,
Nigro
P
,
Thomas
T
,
Le
NT
,
Chang
E
, et al
PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 sumoylation
.
J Cell Biol
2011
;
193
:
867
84
.
10.
Zhang
X
,
Jia
D
,
Liu
H
,
Zhu
N
,
Zhang
W
,
Feng
J
, et al
Identification of 5-iodotubercidin as a genotoxic drug with anti-cancer potential
.
PLoS One
2013
;
7
:
e62527
.
11.
Maddocks
OD
,
Berkers
CR
,
Mason
SM
,
Zheng
L
,
Blyth
K
,
Gottlieb
E
, et al
Serine starvation induces stress and p53-dependent metabolic remodeling in cancer cells
.
Nature
2013
;
493
:
542
6
.
12.
Fujiwara
K
,
Daido
S
,
Yamamoto
A
,
Kobayashi
R
,
Yokoyama
T
,
Aoki
H
, et al
Pivotal role of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in apoptosis and autophagy
.
J Biol Chem
2008
;
283
:
388
97
.
13.
Fotedar
R
,
Brickner
H
,
Saadatmandi
N
,
Rousselle
T
,
Diederich
L
,
Munshi
A
, et al
Effect of p21waf1/cip1 transgene on radiation induced apoptosis in T cells
.
Oncogene
1999
;
18
:
3652
8
.
14.
Mohapatra
P
,
Preet
R
,
Das
D
,
Satapathy
SR
,
Choudhuri
T
,
Wyatt
MD
, et al
Quinacrine-mediated autophagy and apoptosis in colon cancer cells is through a p53- and p21-dependent mechanism
.
Oncol Res
2012
;
20
:
81
91
.