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Carcinogenesis Advance Access originally published online on June 25, 2008
Carcinogenesis 2008 29(10):1878-1884; doi:10.1093/carcin/bgn150
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Sp1 and p73 activate PUMA following serum starvation

Lihua Ming{dagger}, Tsukasa Sakaida{dagger}, 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: +412 623 7786; Fax: +1 412 623 7778; Email: yuj2{at}upmc.edu


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
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 p53-binding sites in the PUMA promoter. Sp1 and p73β 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 p53-independent induction of PUMA following serum starvation to trigger apoptosis in human cancer cells.

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


   Introduction
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 Introduction
Materials and methods
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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), insulin-like 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 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-{kappa}B, 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
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 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
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, {alpha}-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 p73β 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 µg luciferase reporter plasmid and 0.15 µg pCMVβ (Promega, Madison, WI) using LipofectamineTM 2000 reagent (Invitrogen). Luciferase and β-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 β-galactosidase to obtain relative luciferase activity. All reporter experiments were performed in triplicate and repeated three times. In some experiments, 0.4 µg of pCMV-Sp1 and/or 0.2 µg of pcDNA-HA-p73β 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. Approximately 5 x 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 x 106 cells were transfected with p73β expression construct 24 h prior to serum starvation. PCR was performed with primers (5'-GTCGGTCTGTGTACGCATCG-3' and 5'-CCCGCGTGACGCTACGGCCC-3') (13). For each 25 µl 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 p73β 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
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
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).


Figure 1
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  		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 µM) or the transcription inhibitor actinomycin D (Act-D, 10 µg/ml) on PUMA expression were analyzed in HT29 cells by western blotting. {alpha}-Tubulin was used as a loading control.

 
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 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).


Figure 2
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  		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).

 
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 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.


Figure 3
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  		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 [GenBank] ) 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 {alpha}-tubulin were analyzed by western blotting.

 
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 p73β (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 [p73βP292H and p73βS145A (31,32)] did not activate Frag c (Figure 4B).


Figure 4
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  		Fig. 4. p73 is required for PUMA induction following serum starvation. (A) The p73β 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 p73β or the DNA-binding 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 p73β 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 {alpha}-tubulin were analyzed by western blotting.

 
Furthermore, serum starvation or p73β 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 p73β was first transfected into HT29 cells to facilitate its detection. Serum starvation was found to significantly enhance the binding of HA-p73β 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.

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 p73β 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.


Figure 5
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  		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 p73β and Sp1 were immunoprecipitated with a Sp1 antibody or IgG as a control. Western blotting was performed to detect p73β and Sp1. Input represented 10% of the starting material. (C) The 293 cells transfected with p73β were immunoprecipitated with a Sp1 antibody or IgG following serum starvation as in (B). Western blotting was performed to detect p73β and endogenous Sp1.

 
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 p73β 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 jun-N-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 mitogen-activated protein kinase inhibitor PD98059 did not induce PUMA 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.


Figure 6
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  		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 (+serum). The levels of PUMA, total AKT, ERK, phospho-AKT, phospho-ERK, p73 and {alpha}-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.

 
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 following serum starvation, which was accompanied by PUMA induction (Figure 7A and B).


Figure 7
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  		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 {alpha}-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 {alpha}-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.

 
We then determined whether PUMA is required for serum starvation-induced apoptosis in K562 cells, which are susceptible to siRNA-mediated 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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
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 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
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
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 [GenBank] ) to J.Y.; National Institutes of Heath (CA106348 [GenBank] and CA121105 [GenBank] ), American Cancer Society (RSG-07-156-01-CNE) to L.Z.


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
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.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received March 28, 2008; revised June 5, 2008; accepted June 13, 2008.


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W. Qiu, E. B. Carson-Walter, S. F. Kuan, L. Zhang, and J. Yu
PUMA Suppresses Intestinal Tumorigenesis in Mice
Cancer Res., June 15, 2009; 69(12): 4999 - 5006.
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