Carcinogenesis vol.29 no.10 pp.1878–1884, 2008
doi:10.1093/carcin/bgn150
Advance Access publication June 25, 2008
Sp1 and p73 activate PUMA following serum starvation
Lihua Mingy, Tsukasa Sakaiday, Wen Yue, Anupma Jha,
Lin Zhang and Jian Yu
Departments of Pathology, and Pharmacology and Chemical Biology,
University of Pittsburgh Cancer Institute, University of Pittsburgh School of
Medicine, Pittsburgh, PA 15213, USA
To whom correspondence should be addressed. University of Pittsburgh
Cancer Institute, Hillman Cancer Center Research Pavilion, Suite 2.26h, 5117
Centre Avenue, Pittsburgh, PA 15213, USA. Tel: þ412 623 7786;
Fax: þ1 412 623 7778;
Email: [email protected]
p53-upregulated modulator of apoptosis (PUMA) plays an essential role in p53-dependent apoptosis following DNA damage.
PUMA also mediates apoptosis independent of p53. In this study,
we investigated the role and mechanism of PUMA induction in
response to serum starvation in p53-deficient cancer cells. Following serum starvation, the binding of Sp1 to the PUMA promoter
significantly increased, whereas inhibition of Sp1 completely abrogated PUMA induction. p73 was found to be upregulated by
serum starvation and mediate PUMA induction through the p53binding sites in the PUMA promoter. Sp1 and p73b appeared to
cooperatively activate PUMA transcription, which is inhibited by
the phosphoinsitide 3-kinase (PI3K)-protein kinase B (AKT)
pathway. Furthermore, knockdown of PUMA suppressed serum
starvation-induced apoptosis in leukemia cells. Our results
suggest that transcription factors Sp1 and p73 mediate p53independent induction of PUMA following serum starvation to
trigger apoptosis in human cancer cells.
Introduction
Growth factor deprivation triggers apoptosis through p53-independent
mechanisms in some cells (1,2). Many tumor cells activate survival
signaling in the absence of proper growth stimuli, which in turn leads
to suppression of apoptosis and spreading to distant sites. For example,
constitutively active epidermal growth factor receptor (EGFR), insulinlike growth factor-1 (TGF-1) and phosphoinsitide 3-kinase (PI3K)–
protein kinase B (AKT) pathways are among those commonly found
in cancer (3). Cancer cells can become highly dependent on such alterations. As a result, blocking these signals can induce apoptosis mediated
through the mitochondrial pathway, which is prevented by overexpression of the antiapoptotic members of Bcl-2 family of proteins (2,4,5).
The BH3-only subgroup of the Bcl-2 family of proteins is responsible for initiating apoptosis by antagonizing the function of the antiapoptotic members in response to distinct stimuli (6,7). The BH3-only
protein, p53-upregulated modulator of apoptosis (PUMA), was initially identified as a critical mediator of apoptosis induced by the
tumor suppressor p53 and DNA-damaging agents (8,9). PUMA plays
an essential role in p53-dependent and -independent apoptosis in
human cancer cells and mouse cells and mediates apoptosis through
Bax/Bak and the mitochondrial pathway (10–12). PUMA induction
by DNA damage is entirely dependent on an intact p53 and mediated
through the well-defined p53-responsive elements in its promoter
(8,9,13). On the other hand, PUMA is also induced by non-genotoxic
stimuli such as growth factor deprivation. This mode of PUMA induction is independent of p53 but the underlying mechanism is not
well understood (14–16). Several other transcription factors have been
Abbreviations: AKT, protein kinase B; ChIP, chromatin immunoprecipitation;
ERK, extracellular signal-regulated kinase; KO, knockout; PCR, polymerase
chain reaction; PI3K, phosphoinsitide 3-kinase; PUMA, p53-upregulated
modulator of apoptosis; siRNA, small interference RNA.
y
These authors contributed equally to this work.
implicated in regulating PUMA expression, including the p53 family
member p73, E2F1 and FoxO3A (17,18).
Transcription factor Sp1 recognizes GC-rich DNA sequences or
a GC box and is ubiquitously expressed (19,20). Sp1 physically interacts with other transcription factors, including p53, NF-jB, GATA,
YY1, E2F1 and p73 to regulate a wide range of cellular processes and
gene expression in a tissue- and stimulus-specific manner (21–25).
Sp1 was reported to regulate apoptosis in a DNA binding-dependent
manner in some cells (26). The Sp1-binding sites are found in the
promoters of many genes directly involved in apoptosis (27). For
example, Sp1 was reported to mediate the induction of tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL) in histone deacetylase inhibitor-induced apoptosis (28).
In the current study, we investigate the role and mechanism of
PUMA induction following serum starvation in p53-deficient cancer
cells. We identify Sp1 and p73 as critical transcriptional activators of
PUMA following serum starvation and provide a molecular mechanism of serum starvation-induced apoptosis in human cancer cells that
are deficient in p53.
Materials and methods
Cell culture and drug treatments
The human colorectal cancer cell lines HCT116, HT29, DLD1 and SW837,
kidney cell line 293 and leukemia cell lines U937, K562, HL60 and Jurkat
were obtained from American Type Culture Collection (Manassas, VA) and
maintained at 37°C in an atmosphere of 5% CO2 and 95% air. HCT116 cells
with targeted deletion of p53 gene [p53 knockout (KO) cells] were obtained
from Dr Bert Vogelstein (Howard Hughes Medical Institute, the Sidney Kimmel
Comprehensive Cancer Center at Johns Hopkins) (29). All colorectal cancer cell
lines were maintained in McCoy’s 5A medium (Invitrogen, Carlsbad, CA). The
293 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen). The leukemia cell lines were maintained in RPMI (Invitrogen). All media
were supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100
U/ml of penicillin and 100 mg/ml of streptomycin (Invitrogen). Chemotherapeutic agents and inhibitors included phorbol-12-myristate-13-acetate,
PD98059, thapsigargin, brefeldin A and dexamethasone (Calbiochem, San
Diego, CA), cisplatin, adriamycin, wortmannin, mithramycin A, actinomycin
D and cycloheximide (Sigma–Aldrich, St Louis, MO).
Western blotting and antibodies
Western blotting was performed as described previously (10). Antibodies used
in these experiments included those against PUMA (10), p53, Sp1, HA (Santa
Cruz Biotechnology, Santa Cruz, CA), phospho-AKT (Ser 473), total AKT,
phospho-p44/42 extracellular signal-regulated kinase (ERK) (Tyr202/Tyr204),
total p44/42 (Cell Signaling Technology, Danvers, MA), p21, a-tubulin
(Calbiochem) and p73 (Ab-4, Lab Vision, Fremont, CA).
Expression constructs and reporter assay
PUMA promoter-containing fragments were generated by polymerase chain
reaction (PCR) using a previously characterized bacterial artificial chromosome (8) and cloned into the pBV-Luc plasmid. The inserts were verified by
sequencing. The primer sequences are available upon request. The Sp1 expression construct was a kind gift from Dr Robert Tjian (University of California,
Berkley, CA). The p73b expression constructs were from Drs Carol Prives
(Columbia University, New York, NY) (WT and S145A) and William Kaelin
(Harvard University, Cambridge, MA) (R292H). Reporter assays were performed as described (8). In brief, transfections were performed in 12-well
plates using 0.3 lg luciferase reporter plasmid and 0.15 lg pCMVb (Promega,
Madison, WI) using LipofectamineTM 2000 reagent (Invitrogen). Luciferase
and b-galactosidase activities were assessed 24–48 h following transfection
using reagents from Promega and ICN Biomedicals (Costa Mesa, CA), respectively. The luciferase activities were normalized to b-galactosidase to
obtain relative luciferase activity. All reporter experiments were performed
in triplicate and repeated three times. In some experiments, 0.4 lg of
pCMV-Sp1 and/or 0.2 lg of pcDNA-HA-p73b were used in each transfection.
Chromatin immunoprecipitation assay
The assay was performed using a kit from Upstate Biotechnology (Charlottesville, VA) following the manufacturer’s instructions with minor modifications.
Ó The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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PUMA is regulated by Sp1 and p73
Approximately 5 106 cells were used in each chromatin immunoprecipitation
(ChIP) assay. The antibodies used included anti-Sp1, HA (Santa Cruz Biotechnology), p21 and rabbit IgG (R&D systems, Minneapolis, MN). For Sp1-ChIP,
PCR was performed using primers (5#-GACTTTGTGGACCCTGGAACG-3#
and 5#-CTAGCCCAAGGCAAGGAGGACC-3#). For p73-ChIP, 5 106
cells were transfected with p73b expression construct 24 h prior to serum
starvation. PCR was performed with primers (5#-GTCGGTCTGTGTACGCATCG-3# and 5#-CCCGCGTGACGCTACGGCCC-3#) (13). For each 25 ll
reaction, 10 pmol of the primers was used and 30 cycles of PCR were performed. Each experiment was done at least twice with similar results.
Immunoprecipitation
Immunoprecipitation was carried out as described (8). In brief, HT29 cells
were cotransfected with Sp1 expression construct and p73b expression construct. Anti-Sp1 (E3, Santa Cruz Biotechnology) was used for the immunoprecipitation with mouse IgG (R&D Systems) as a control.
Small interference RNA transfection
The cells at 50–60% confluency or 400 000 cells (K562 cells) were transfected
with the p73 or PUMA small interference RNA (siRNA) duplexes (Dharmacon,
Lafayette, CO) with Lipofectamine 2000 following the manufacturer’s instructions. The target complementary DNA sequences of p73 and PUMA siRNA
duplexes are 5#-CGGATTCCAGCATGGACGT-3# (p73), 5#-ACCTCAACGCACAGTACGA-3# (PUMA 721) and 5#-ACTCAACGCACAGTACGA-3#
(PUMA 1559), respectively. The LaminA/C or scrambled siRNA (Dharmacon)
was used as a control in these experiments. Twenty-four hours after transfection, the cells were treated with serum starvation for 24 or 48 h and harvested
for protein or apoptosis analysis.
Analysis of apoptosis
Apoptosis was assessed through microscopic visualization of condensed chromatin and micronucleation or by flow cytometry with Cytomation Summit
software as described previously (30).
Bioinformatic and statistical analysis
The bioinformatics tools for identification of potential transcription factor-binding
sites include transcription factor (TF) search (http://www.cbrc.jp/research/db/
TFSEARCH.html) and DNAsis (Hitachi Software Engineering America Ltd,
South San Francisco, CA). The means ± 1 SDs are displayed in the figures.
Results
PUMA is induced by serum starvation in p53-deficient cells
To study p53-independent regulation of PUMA, we analyzed PUMA
protein levels in HCT116 cells with targeted deletion in the p53 gene
(p53 KO cells) (29), following the treatments of several agents that
promote apoptosis but do not damage DNA. Several classes of proapoptotic stimuli were used, including serum starvation, protein kinase C
activator phorbol-12-myristate-13-acetate, mitogen-activated protein
kinase inhibitor PD98059, endoplasmic reticulum (ER) stressors thapsigargin and brefeldin A and glucocorticoid dexamethasone. As expected, DNA-damaging agent cisplatin induced PUMA in parental
HCT116 cells containing the intact p53 gene but not in p53 KO cells
(Figure 1A). Serum starvation and two ER stressors induced PUMA in
p53 KO cells at levels similar to that found in parental HCT116 cells
treated with cisplatin. In response to these non-genotoxic agents, the
patterns of induction of the cell cycle regulator p21 were different
from those of PUMA, suggesting that their expression is regulated by
distinct mechanisms in p53 KO cells (Figure 1A).
Serum starvation induced PUMA in four other colon cancer cell
lines with mutant p53, including DLD1, SW480, SW837 and HT29
(Figure 1B). PUMA induction was already evident within 9 h and
continued to increase until 24 h following serum starvation in HT29
cells (Figure 1C). No induction was observed in cells treated with the
transcription inhibitor actinomycin D or the translation inhibitor cycloheximide (Figure 1C). These results indicate that serum starvation
might activate PUMA transcription through a p53-independent
manner.
Identification of the regions in the PUMA promoter that mediate
p53-independent transcription
To identify potential DNA elements responsible for PUMA transcription following serum starvation, an 2 kb fragment of the PUMA
Fig. 1. p53-independent induction of PUMA by serum starvation. (A)
Parental HCT116 cells and isogenic p53 KO cells were subjected to the
indicated treatments for 24 h. The levels of p53, PUMA and p21 were
analyzed by western blotting. (B) The expression of PUMA was analyzed by
western blotting in indicated colon cancer cell lines with mutant p53. (C) The
effects of translation inhibitor cycloheximide (CHX, 2 lM) or the
transcription inhibitor actinomycin D (Act-D, 10 lg/ml) on PUMA
expression were analyzed in HT29 cells by western blotting. a-Tubulin was
used as a loading control.
promoter (Frag E) and four smaller fragments in this region [Frag
A (8), B–D, each containing 500 bp of DNA] were cloned into
a low-background reporter construct pBV-Luc (Figure 2A). The activities of these reporter constructs were tested in p53 KO HCT116
cells and HT29 cells. Only the reporters containing full-length Frag E
or the proximal Frag A were found to have measurable activities in the
absence of serum (Figure 2B).
To further identify the elements that mediate PUMA transactivation
within Frag A, we tested various smaller fragments within this region
in reporter assays, including the fragments a–e (100 bp each), abc
(300 bp) and de (200 bp) (Figure 2C). The fragments A, abc, de, c
and d were found to be significantly activated by serum starvation in
HT29 and p53 KO cells (Figure 2D and data not shown). These results
indicate that the proximal region of the PUMA promoter (nucleotide
295/95, Figure 2C and D) is largely responsible for its transcription following serum starvation, and Frag c and Frag d might contain
binding sites for specific transcription factors.
Identification of Sp1 as a transcriptional activator of PUMA following
serum starvation
We analyzed the proximal PUMA promoter region using bioinformatics tools including TF search and DNAsis and identified several
Sp1 sites in Frag d and Frag c (Figures 2C and 3A). The reporters
containing Frag b, Frag c and Frag d were activated by Sp1. Frag
d showed the highest activities among them and contains multiple Sp1
sites (Figure 3B). ChIP assay was used to analyze the binding of Sp1
to the PUMA promoter in HT29 cells before and after serum starvation. The recruitment of Sp1 to the PUMA promoter is greatly enhanced by serum starvation at 24 h (Figure 3C). In contrast, no
difference in the binding was detected with the control p21 antibody
or isotype-matched IgG (Figure 3C). The binding of nuclear extracts
to Sp1 sites within the PUMA promoter was induced by serum starvation as well (data not shown). Finally, the Sp1 inhibitor mithramycin A was found to completely suppress serum starvation-induced
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L.Ming et al.
Fig. 2. Identification of the DNA elements in the PUMA promoter involved in p53-independent transcription. (A) The schematic representation of the various
fragments (Frag A–E) in the PUMA promoter cloned into luciferase reporter constructs. (B) The normalized relative luciferase activities (RLAs) of the indicated
reporters in HT29 and p53 KO cells were plotted. That of the empty vector was defined as 1. (C) The schematic representation of various fragments (Frag a–e, abc
and de) in the proximal PUMA promoter Frag A. The two p53-binding sites (BS1 and BS2) and several Sp1 sites were indicated. (D) Relative luciferase activities
of the indicated reporters in HT29 cells cultured with or without serum were measured as in (B).
PUMA expression (Figure 3D). These experiments indicate that Sp1
can directly bind to the PUMA promoter to drive its transcription
following serum starvation in p53-deficient cells.
Identification of p73 as a transcriptional activator of PUMA following
serum starvation
Frag c contains two previously identified p53-binding sites and
showed serum-induced activities in reporter assays in p53 KO or deficient cells (Figure 2C and D) (8). The p53 family member p73 was
reported to activate p53 target genes, including PUMA (17). We therefore tested whether p73 could mediate PUMA induction following
serum starvation. We found that only the reporters containing the two
p53-binding sites, such as Frag abc and Frag c, were significantly
activated by expression of p73b (Figure 4A). The Frag abc was less
active compared with Frag c, suggesting that other cis elements in
Frag abc might suppress PUMA transcription. Two DNA-binding
mutants of p73 [p73bP292H and p73bS145A (31,32)] did not activate
Frag c (Figure 4B).
Furthermore, serum starvation or p73b only activated the PUMA
reporter containing synthetic p53-binding sites of BS2 but not BS1 (8)
in p53 KO cells, and mutations of the two critical nucleotides in the
p53-binding sites completely abrogated this activity (Figure 4C and
D). The binding of p73 to the PUMA promoter before and after serum
starvation was further analyzed by ChIP in HT29 cells. As several p73
antibodies failed to precipitate the endogenous p73, HA-tagged p73b
was first transfected into HT29 cells to facilitate its detection. Serum
starvation was found to significantly enhance the binding of HA-p73b
to the PUMA promoter (Figure 4E). Knockdown of p73 by siRNA
impaired serum starvation-induced PUMA expression (Figure 4F).
These data suggest that p73 activates PUMA transcription following
serum starvation through the p53-binding sites.
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Sp1 cooperates with p73 to activate PUMA transcription
Sp1 was reported to cooperate with p53 to regulate p21 and PUMA
following DNA damage and was found to interact with p73 in vitro
(24,25,33). Sp1 might cooperate with p73 to induce PUMA transcription as their binding sites are in close proximity (Figure 2C).
To test this, Sp1 and p73 expression plasmids were transfected either
alone or in combination into p53 KO cells along with various PUMA
reporter constructs. Coexpression of Sp1 and p73 enhanced the activities of reporters containing Frag abc and Frag c compared with
either one alone (Figure 5A). Furthermore, we found that Sp1 and
p73b can form a complex in 293 cells when expressed together or
when only p73 was overexpressed (Figure 5B and C). However, we
did not observe an obvious increase in their association following
serum starvation in this system (data not shown). These data suggest
that Sp1 and p73 can interact and cooperatively activate PUMA
transcription.
The PI3K–AKT pathway suppresses the expression of PUMA
The PI3K–AKT pathway promotes survival while suppressing apoptosis and is a well-established downstream effector of the growth
factor signaling (3). We wondered whether the levels of p73 or Sp1 are
affected by the PI3K–AKT pathway. The expression of PUMA and
p73b was significantly suppressed by serum within 24 h in HT29 cells
but not that of Sp1 (Figure 6A and B). Interestingly, serum stimulation
induced phosphorylation of AKT and ERK but not that of p38 and junN-terminal kinase (JNK) (Figure 6A and data not shown). AKT phosphorylation was inversely correlated with PUMA levels (Figure 6B).
The PI3K inhibitor wortmanin significantly elevated PUMA levels in
the presence of serum in HT29, DLD1 and SW837 cells while inhibiting AKT phosphorylation (Figure 6C). In contrast, the mitogenactivated protein kinase inhibitor PD98059 did not induce PUMA
PUMA is regulated by Sp1 and p73
lowing serum starvation, which was accompanied by PUMA induction (Figure 7A and B).
We then determined whether PUMA is required for serum starvation-induced apoptosis in K562 cells, which are susceptible to siRNAmediated gene silencing by liposome-mediated transfection (Figure
7C). Knockdown of PUMA significantly blocked the accumulation of
cells with sub-G1 DNA content following serum starvation (Figure
7D). PUMA siRNA virtually blocked the apoptosis induced by serum
starvation measured by nuclear fragmentation assays (Figure 7E).
These data indicate that PUMA plays an important role in serum
starvation-induced apoptosis in leukemia cells, and this function does
not require p53.
Discussion
Fig. 3. Sp1 as a transcriptional activator of PUMA. (A) Schematic
representation of the Sp1 sites identified by bioinformatics analysis (upper
panel). Predicted high-scoring Sp1 sites (.86) in Frag c–e were underlined
in the lower panel. Additional overlapping Sp1 sites contained in
NT 351-400 (GenBank entry AF332559) were not indicated. (B) The Sp1
expression plasmid or the control vector was transfected with the indicated
PUMA reporter into p53 KO cells. The normalized luciferase activities were
plotted. The basal activity of Frag a was defined as 1. (C) ChIP assay was
used to characterize the recruitment of Sp1 to the PUMA promoter in HT29
cells. Inputs corresponded to 1% of the total chromatin extract used in
immunoprecipitation. The p21 antibody and isotype-matched IgG were
used as controls. (D) HT29 cells were subjected to the indicated treatments
for 24 h. The levels of PUMA and a-tubulin were analyzed by western
blotting.
significantly in p53 KO or HT29 cells (Figure 1A and data not shown).
These results suggest that the PI3K–AKT pathway suppresses PUMA
following the activation of growth factor signaling, perhaps through
the downregulation of p73.
Knockdown of PUMA by siRNA inhibits serum starvation-induced
apoptosis
The genetic and molecular manipulation in colon cancer cell lines
greatly facilitated our analysis on PUMA transcriptional regulation
following serum starvation. However, these cells are resistant to
serum starvation-induced apoptosis, perhaps due to high levels of
cyclin-dependent kinase inhibitor p21 and antiapoptotic protein Bcl-xL
(10,34). Several leukemia cell lines with either null or mutant p53,
including K562, U937, HL60 and Jurkat, underwent apoptosis fol-
In the current study, we found that PUMA plays an important role in
the apoptotic response to serum starvation in human cancer cells
through a p53-independent mechanism. Sp1 and p73 bind to distinct
sites in the PUMA promoter and cooperatively activate PUMA transcription following serum starvation. Growth factor signaling suppresses the expression of PUMA and p73, but not that of Sp1,
through the PI3K–AKT pathway. Our findings have several important
implications in the understanding of apoptosis regulation in human
cancer cells.
In normal cells, lack of growth factors can trigger apoptosis, which
serves as an effective safeguard mechanism against unscheduled proliferation. Cancer cells frequently develop mechanisms to overcome
this barrier (3,35). Our study suggests that constitutive PI3K–AKT
pathway, commonly found in cancer, can suppress apoptosis by inhibiting PUMA expression (Figures 1, 6 and 7). Our finding is consistent with previous findings that PUMA plays an important role in
apoptosis induced by cytokine and growth factor withdrawal in mouse
hematopoetic and lymphoid cells (15,16,18,36,37), whereas growth
factors, such as IGF-1 and epidermal growth factor, suppress the
expression of PUMA (14). Therefore, inhibiting PUMA expression
might be a common mechanism by which deregulated growth factor
signaling suppresses apoptosis.
PUMA is a major mediator of DNA damage-induced apoptosis
through p53-dependent transcription, but little is known how its expression is regulated by non-genotoxic stresses (38). Our data indicate
that Sp1 and p73 are important activators of PUMA transcription
following serum starvation (Figures 3, 4 and 6). The PUMA promoter
contains several Sp1- and p53-binding sites nearby that are known to
facilitate the formation of protein complexes and transcriptional synergism (39,40). This might be enhanced by increased p73 levels or
phosphorylation as reported previously in other cell types (32,41). It
remains to be determined whether the recruitment of Sp1 and p73 to
the PUMA promoter is dependent on each other. Taken together, our
observations are consistent with the notion that p73 might substitute
for p53 to activate its effectors to provide some protection against
neoplastic transformation in p53-deficient cells (25,42).
Targeted deletion of PUMA has little effects on physiological apoptosis but confers a remarkable degree of resistance to stress-induced
apoptosis in a wide variety of cell types, suggesting that PUMA expression is tightly controlled to avoid triggering unwanted apoptosis
in unstressed cells (11,43). Our data provide an explanation for such
a regulation (8). The smaller fragments containing 100 nucleotides
of the proximal PUMA promoter often showed more dramatic activation by Sp1 or p73 compared with larger fragments containing several
100 nucleotides (Figures 3B and 4A). This suggests that transcriptional repression might be responsible for the low basal levels of
PUMA in unstressed cells. One possibility is that the high GC content
of the PUMA promoter favors the formation of secondary structures
that limit the accessibility of the transcriptional machinery or recruit
transcriptional repressors or chromatin-modifying proteins to prevent
active transcription (44,45). The current data support a model in
which p53 induces changes in the chromatin that permits PUMA
transcription following DNA damage (13). It is conceivable that
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L.Ming et al.
Fig. 4. p73 is required for PUMA induction following serum starvation. (A) The p73b expression plasmid was transfected with the indicated PUMA reporters into
p53 KO cells. The normalized luciferase activities were plotted with that of Frag e defined as 1. (B) The expression constructs of the wild-type p73b or the DNAbinding mutants R292H and S145A were cotransfected with the Frag c reporter. The normalized luciferase activities were plotted with that of the empty expression
vector defined as 1. (C) The synthetic reporters containing either four copies of the wild-type or mutant p53-binding sites (BS1 or BS2) in the PUMA promoter
were transfected into the p53 KO cells. The ratio of relative luciferase activities in cells cultured with or without serum was plotted with that of pBV-Luc defined as
1. (D) The p73b expression plasmid was transfected with the indicated synthetic PUMA reporters as in (C) into p53 KO cells. The normalized luciferase activities
were plotted with that of pBV-Luc defined as 1. (E) ChIP assay was used to characterize the recruitment of p73 to the PUMA promoter upon serum starvation.
HT29 cells were transfected with p73 expression vector followed by serum starvation for 24 h. Inputs corresponded to 1% of the total chromatin extract used in
immunoprecipitation. IgG was used as a control. (F) The p73 siRNA or control siRNA (Lamin) was transfected into DLD1 cells for 24 h followed by the indicated
treatments for 24 h. The levels of PUMA, p73 and a-tubulin were analyzed by western blotting.
Fig. 5. p73 and Sp1 cooperate to activate PUMA transcription. (A) The indicated PUMA reporter constructs were cotransfected into the p53 KO cells with the
expression constructs of Sp1, p73 or both. The relative luciferase activities were analyzed 24 h following transfection. The activity of Frag e was defined as 1. (B)
The 293 cells cotransfected with p73b and Sp1 were immunoprecipitated with a Sp1 antibody or IgG as a control. Western blotting was performed to detect p73b
and Sp1. Input represented 10% of the starting material. (C) The 293 cells transfected with p73b were immunoprecipitated with a Sp1 antibody or IgG following
serum starvation as in (B). Western blotting was performed to detect p73b and endogenous Sp1.
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PUMA is regulated by Sp1 and p73
binding of p73 and Sp1 to the PUMA promoter induces similar
changes following serum starvation.
Mutation or gene silencing by promoter methylation in p53 targets
is extremely rare in human cancer, including PUMA (11). This is
perhaps not surprising as dysfunctional p53 or constitutive PI3K–
AKT signaling can prevent PUMA induction, which is found in most
if not all cancer. Earlier studies from us and others have demonstrated
that elevated PUMA expression is profoundly toxic to cancer cells and
sensitizes them to chemotherapy or radiation (8,9,46–48). Therefore,
defining p53-independent mechanisms of PUMA activation may
facilitate the development of new therapeutic strategies targeting human tumors by overcoming their intrinsic or acquired resistance to
apoptosis.
Funding
Flight Attendant Medical Research Institute, Alliance for Cancer
Gene Therapy, University of Pittsburgh Cancer Institute Head and
Neck SPORE Career Development Award (1 P50 CA097190) to
J.Y.; National Institutes of Heath (CA106348 and CA121105),
American Cancer Society (RSG-07-156-01-CNE) to L.Z.
Fig. 6. Growth factor signaling suppresses PUMA through the PI3K–AKT
pathway. (A) HT29 cells were serum starved for 24 h (serum) and 10%
serum was added to the medium for 24 h (þserum). The levels of PUMA,
total AKT, ERK, phospho-AKT, phospho-ERK, p73 and a-tubulin were
analyzed by western blotting. (B) HT29 cells were serum starved for 24 h and
10% serum was added back to the medium for the indicated time. The
indicated proteins were analyzed by western blotting as in (A). (C) The
effects of wortmannin on the indicated proteins were analyzed by western
blotting.
Acknowledgements
The authors would like to thank other members of our laboratories for helpful
discussions and comments. We would like to thank Drs Bert Vogelstein, Robert
Tjian, William Kaelin, Carol Prives and Daniel E.Johnson for providing reagents. We would like to thank Drs Richard Steinmen, Ruth Modzelewski and
Pamela Hershberger for critical reading. L.Z. is a V Scholar.
Conflict of Interest Statement: None declared.
Fig. 7. PUMA is involved in serum starvation-induced apoptosis in leukemia cells. (A) The indicated leukemia cell lines were serum starved for 48 h and analyzed
for apoptosis by flow cytometry. The fractions of cells in sub-G1 (apoptotic cells) were indicated. (B) The expression of PUMA and a-tubulin was analyzed by
western blotting. (C) K562 cells were transfected with scrambled or PUMA siRNA for 18 h and cultured with or without serum for 48 h. The levels of PUMA and
a-tubulin were analyzed by western blotting. (D) K562 cells were transfected and treated as in (C) and analyzed as in (A). (E) The cells in (D) were analyzed by
nuclear fragmentation assay.
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Received March 28, 2008; revised June 5, 2008; accepted June 13, 2008