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Cancer Therapy: Preclinical
PUMA Sensitizes Lung Cancer Cells to Chemotherapeutic
Agents and Irradiation
Jian Yu,1,3 Wen Yue,2,3 Bin Wu,2,3 and Lin Zhang2,3
Abstract
Purpose: Lung cancer, the leading cause of cancer mortality worldwide, is often diagnosed
at late stages and responds poorly to conventional therapies, including chemotherapy and irradiation. A great majority of lung tumors are defective in the p53 pathway, which plays an important
role in regulating apoptotic response to anticancer agents. PUMA was recently identified as an
essential mediator of DNA damage ^ induced and p53-dependent apoptosis. In this study, we
investigated whether the regulation of PUMA by anticancer agents is abrogated in lung cancer
cells and whether PUMA expression suppresses growth of lung cancer cells and/or sensitizes
lung cancer cells to chemotherapeutic agents and irradiation through induction of apoptosis.
Experimental Designs: The expression of PUMA was examined in lung cancer cells with
different p53 status treated with chemotherapeutic agents. An adenovirus expressing PUMA
(Ad-PUMA), alone or in combination with chemotherapeutic agents or g-irradiation, was used to
treat lung cancer cells. The growth inhibitory and apoptotic effects of PUMA in vitro and in vivo
were examined. The mechanisms of PUMA-mediated growth suppression and apoptosis were
investigated through analysis of caspase activation and release of mitochondrial apoptogenic
proteins.The cytotoxicities of PUMA on cancer and normal/nontransformed cells were compared.
The efficacy of PUMA and p53 in suppressing the growth of lung cancer cells was also compared.
Results: We showed that the induction of PUMA by chemotherapeutic agents is abolished in
p53-deficient lung cancer cells. PUMA expression resulted in potent growth suppression of lung
cancer cells and suppressed xenograft tumor growth in vivo through induction of apoptosis.
Low dose of Ad-PUMA significantly sensitized lung cancer cells to chemotherapeutic agents
and g-irradiation through induction of apoptosis.The effects of PUMA are mediated by enhanced
caspase activation and release of cytochrome c and apoptosis-inducing factor into the cytosol.
Furthermore, PUMA seems to be selectively toxic to cancer cells and more efficient than p53 in
suppressing lung cancer cell growth.
Conclusions: Our findings indicate that PUMA is an important modulator of therapeutic
responses of lung cancer cells and is potentially useful as a sensitizer in lung cancer therapy.
Lung cancer is the leading cause of cancer mortality worldwide
(1). The current treatment options for patients with advanced
disease are limited to chemotherapy and radiation therapy,
which unfortunately produce a low rate of response and virtually
no cure. The 5-year survival rate, currently at 14%, has only been
Authors’ Affiliations: Departments of 1Pathology and 2Pharmacology, and
3
University of Pittsburgh Cancer Institute, University of Pittsburgh School of
Medicine, Pittsburgh, Pennsylvania
Received 11/9/05; revised 1/17/06; accepted 2/27/06.
Grant support: The Flight Attendant Medical Research Institute, the Alliance for
Cancer Gene Therapy, the Hillman Foundation (J. Yu), NIH grant CA106348, the
Edward Mallinckrodt Jr. Foundation, the Elsa U. Pardee Foundation, the General
Motors Cancer Research Foundation, the V Foundation for Cancer Research,
University of Pittsburgh Cancer Institute Lung Specialized Program of Research
Excellence career development award, and the Outstanding Overseas Young
Scholar Award from the Chinese Natural Science Foundation (L. Zhang).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Jian Yu, University of Pittsburgh Cancer Institute, Hillman
Cancer Center Research Pavilion, Suite 2.26H, 5117 Centre Avenue, Pittsburgh, PA
15213. Phone: 412-623-7786; Fax: 412-623-7778; E-mail: yuj2@ upmc.edu.
F 2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-05-2429
Clin Cancer Res 2006;12(9) May 1, 2006
marginally improved in the last two decades despite the
development of new therapeutic agents and improved patient
care. Novel therapeutic interventions are critical for improvement of the survival and prognosis of lung cancer patients.
Cancer development is a multistage process which involves a
number of genetic and epigenetic changes in the genes
controlling cell survival, cell death, cell-cell communication,
cell-microenvironment interactions, and angiogenesis (2, 3). It
has become increasingly clear that abnormalities in cell death
pathways play an important role in tumorigenesis and the
development of resistance to chemotherapy and radiation
therapy (4, 5). Most of the agents used in cancer therapy
directly or indirectly damage DNA and induce apoptosis.
Defects in the apoptotic machinery can lead to multidrug
resistance (6). Therapeutic manipulation of the apoptotic
pathways has become an attractive avenue to improve the
clinical response of lung cancer patients (7 – 9).
Defective p53 pathway is one of the most common
signatures of human cancer (2). p53 mutations occur in
f50% of non – small-cell lung cancers and >70% of small-cell
lung cancers (10). A major physiologic function of p53 is to kill
damaged or stressed cells through induction of apoptosis (11).
p53 induces apoptosis by transactivation of its downstream
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Chemosensitization and Radiosensitization by PUMA
apoptotic regulators. p53 mutations in cancer cells almost
invariably abolish this activity, indicating that the apoptotic
function of p53 is critical for its tumor suppressor activity (11).
Restoration of the p53 pathway by activating p53 itself or p53
downstream targets has been explored to improve efficacy of
anticancer therapies (12).
PUMA was recently identified as a BH3-only Bcl-2 family
protein and an essential mediator of p53-dependent and
p53-independent apoptosis (13). PUMA is induced by DNAdamaging agents in a p53-dependent fashion and by nongenotoxic agents independent of p53 (14 – 16). Expression of
PUMA rapidly kills a variety of human cancer cells (14, 15).
PUMA is localized in the mitochondria and induces apoptosis by activating caspases through mitochondrial dysfunction (14, 15, 17). PUMA functions through other Bcl-2
family members, such as Bax, Bcl-2, and Bcl-XL. Deletion of
PUMA in human cancer cells attenuated apoptotic response
to p53, DNA-damaging agents, and hypoxia (17). PUMAknockout mice recapitulated several major apoptotic deficiencies observed in p53-knockout mice (18, 19). However,
the role of PUMA in therapeutic responses in lung cancer
cells remains unclear.
In this study, we examined the regulation of PUMA by
commonly used anticancer agents in lung cancer cells. We
found that p53 mutations abolish the induction of PUMA
by these agents. We also showed that PUMA is a potent and
selective inducer of apoptosis in lung cancer cells and that
expression of PUMA enhances the therapeutic responses of
lung cancer cells to chemotherapeutic agents and g-irradiation.
Materials and Methods
Cell culture and drug treatment. The cell lines used in the study
were from American Type Culture Collection (Manassas, VA), except
for the lung cancer cell lines 273T and 201T, which were from the
University of Pittsburgh Cancer Institute lung cancer program, and the
human primary fibroblast cell line WI-38, which was obtained from
the Coriell Institute (Camden, NJ). All cell lines were maintained at
37jC and 5% CO2. Cell culture media included RPMI 1640
(Mediatech, Herndon, VA) for all lung cancer cell lines, McCoy’s 5A
(Invitrogen, Carlsbad, CA) for colorectal cancer cell lines HCT116 and
DLD1, DMEM (Mediatech) for 293 and 911 cells, and EMEM
(Invitrogen) for WI-38. The cell culture media were supplemented
with 10% fetal bovine serum (HyClone, Logan, UT), 100 units/mL
penicillin, and 100 Ag/mL streptomycin (Invitrogen), except for
EMEM, which was supplemented with 20% fetal bovine serum.
Transfection was done with Lipofectamine 2000 (Invitrogen) following the instructions of the manufacturer. Reporter assays were carried
out as previously described (14).
The anticancer drugs used in the study, including Adriamycin,
oxaliplatin, cisplatin, 5-fluorouracil (5-FU), etoposide, and Taxol, were
purchased from Sigma (St. Louis, MO). Caspase inhibitors, including
the pan-caspase inhibitor Z-VAD-fmk, caspase-3 inhibitor Z-DEVDfmk, caspase-8 inhibitor Z-IETD-fmk, and caspase-9 inhibitor ZLEHD-fmk, were purchased from R&D Systems (Minneapolis, MN).
All drugs and inhibitors were dissolved in DMSO and diluted to
appropriate concentrations with cell culture media. Some cells were
exposed to g-irradiation at 8 Gy. For combination treatments, cells
were infected with adenoviruses for 16 to 18 hours before drug
treatment or g-irradiation.
Constructs and adenoviruses. The expression constructs used in
the study included those expressing wild-type and different forms of
mutant p53, including V143A, R175H, R249S, and R273H (20), and
constructs expressing NH2-terminal green fluorescent protein (GFP) –
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Fig. 1. p53-dependent activation of PUMA by chemotherapeutics in lung cancer
cells. A , eight lung cancer cell lines with different p53 status were treated with
Adriamycin (0.2 Ag/mL) for 24 hours and analyzed for the expression of PUMA,
p21, and p53 by Western blotting. a-Tubulin was used as a loading control.
B, reverse transcription-PCR was done for PUMA (25 and 35 cycles) and the
control GAPDH (20 cycles) using the cDNA prepared from cells with or without
Adriamycin treatment. MW, molecular weight marker; Bl, no-template PCR control.
C, PUMA luciferase reporters were cotransfected with wild-type (Wt) or mutant
p53, along with the control pCMVh plasmid, into H1299 cells. Frag 1 (f500 bp)
and Frag 2 (f200 bp) are previously described reporters with and without the
p53-binding sites, respectively (14). The relative luciferase activities normalized to
the h-galactosidase activity were determined 48 hours following transfection.
tagged proteins, including wild-type PUMA, PUMA lacking the BH3
domain (DBH3), or the mitochondrial targeting sequence of PUMA
(C43; ref. 17). For reporter assays, the previously described PUMA
luciferase reporter constructs Frag 1 and Frag 2 were cotransfected with
the h-galactosidase reporter pCMVh (Promega; ref. 14).
The recombinant adenoviruses Ad-PUMA, Ad-DBH3, and Ad-p53
were constructed using the Ad-Easy system as previously described
(17, 21). High-titer viruses were produced in 293 cells and purified by
CsCl2 gradient ultracentrifugation (22).
Apoptosis and growth assays. After treatment, attached and floating
cells were harvested and analyzed for apoptosis by nuclear staining with
propidium iodide (23). A minimum of 300 cells were analyzed for each
treatment. The propidium iodide – stained cells were analyzed by flow
cytometry to determine the fraction of sub-G1 cells. Apoptosis was
also analyzed by staining cells with Annexin V-Alexa 594 (Molecular
Probes, Carlsbad, CA), counterstaining with 4V,6-diamidino-2-phenylindole, followed by flow cytometry. Cell growth was measured by
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3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay in 96-well plates (2,500 cells per
well) using the CellTiter 96 AQueous One Solution (Promega, Madison,
WI) following the instructions of the manufacturer. A 490 nm was
measured using a Victor III (Perkin-Elmer/Wallace) plate reader. Each
experiment was done in triplicate and repeated at least twice.
Western blotting and antibodies. Western blotting was done as
previously described (24). The antibodies used for Western blotting
included rabbit polyclonal antibodies against PUMA (17), caspase 9
(Cell Signaling Technology, Danvers, MA), caspase 3 (Stressgen, San
Diego, CA), and HA (Santa Cruz Biotechnology, Santa Cruz, CA);
monoclonal antibodies against a-tubulin (Oncogene Sciences, San
Diego, CA), p21 (Oncogene Sciences), cytochrome c (BD Biosciences,
San Jose, CA), cytochrome oxidase subunit IV (Molecular Probes),
caspase 8 (Cell Signaling Technology), and p53 (DO1, Santa Cruz
Biotechnology); and a goat antibody against apoptosis-inducing factor
(Santa Cruz Biotechnology).
Cellular fractionation. Floating and attached cells were harvested
from two 75-cm2 (T75) flasks by centrifugation, resuspended in
homogenization buffer [0.25 mol/L sucrose, 10 mmol/L HEPES
(pH 7.4), and 1 mmol/L EGTA], and subjected to 40 strokes of
homogenization on ice in a 2-mL Dounce homogenizer. The
homogenates were centrifuged at 1,000 g at 4jC for 10 minutes
to pellet nuclei and unbroken cells. The supernatant was subsequently
centrifuged at 10,000 g at 4jC for 15 minutes to obtain cytosolic
(supernatant) and mitochondrial (pellet) fractions.
Reverse transcription-PCR. Total RNA was isolated using the
RNAgents Total RNA Isolations System (Promega) according to the
instructions of the manufacturer. First-strand cDNA was synthesized
using Superscript II reverse transcriptase (Invitrogen). Reverse
transcription-PCR was done to amplify PUMA using the cycle
conditions previously described (14). The primers used to amplify
PUMA included 5-tcctcagccctcgctctcgc-3Vand 5V-ccgatgctgagtccatcagc-3V.
The primers for amplifying the control glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were 5V-ctcagacaccatggggaaggtga-3V and 5Vatgatcttgaggctgttgtcata-3V.
Xenograft tumors and tissue staining. All animal experiments were
approved by the Institutional Animal Care and Use Committee at
the University of Pittsburgh. Xenograft tumors were established by
s.c. injection of 4 106 A549 or H1299 cells into both flanks of
5- to 6-week-old female athymic nude mice (Harlan, Indianapolis,
IN). Tumor treatment was initiated by injecting each tumor
(f50-100 mm3) with Ad-PUMA or Ad-DBH3 at 5 108 plaqueforming units in 100 AL of PBS. Each treatment was repeated
twice. Tumor growth was monitored thrice a week by calipers to
calculate tumor volumes according to the formula (length width2) / 2.
Terminal deoxyribonucleotidyl transferase – mediated dUTP nick end
labeling staining on frozen sections was done using recombinant
terminal transferase (Roche, Indianapolis, IN) and dUTP-Alexa 594
(Molecular Probes) according to the instructions of the manufacturers
and counterstained by 4V,6-diamidino-2-phenylindole. PUMA expression in frozen sections was determined by fluorescence microscopic
analysis of GFP. Frozen sections were also analyzed by H&E staining. All
images were acquired with a Nikon TS800 fluorescence microscope
using SPOT camera imaging software.
Statistical analysis. Statistical analysis was done using GraphPad
Prism IV software. P values were calculated by Student’s t test or twoway ANOVA. The mean F SD were displayed in the figures.
Results
PUMA Induction by chemotherapeutic agents is abolished in
p53-deficient lung cancer cells. PUMA is normally expressed at
low levels in human tissues but can be induced by p53 or DNAdamaging agents (14, 15). To investigate the regulation of
Clin Cancer Res 2006;12(9) May 1, 2006
PUMA by anticancer agents in lung cancer cells, eight lung
cancer cell lines with different p53 status were treated with
Adriamycin (0.2 Ag/mL), a DNA-damaging agent producing
double-stranded DNA breaks and a chemotherapeutic drug
commonly used for treating lung cancer (25). The expression of
PUMA and p21, which mediate p53-dependent apoptosis and
cell-cycle arrest, respectively, were analyzed (11). Both PUMA
and p21 were found to be induced by Adriamycin in the wildtype p53 cell lines A549 and 128.88T, but not in the p53mutant cell lines DMS53, 201T, 273T, and H1752 and the
p53-null cell lines H1299 and Calu 1 (Fig. 1A). PUMA was also
found to be induced only in the wild-type p53 lung cancer cells
by another chemotherapeutic agent, 5-FU (50 Ag/mL), and
g-irradiation (8 Gy; data not shown).
To determine whether the deficiency in PUMA induction
is at the mRNA level, semiquantitative reverse transcriptionPCR was used to examine PUMA in cells with and without
Adriamycin treatment. PUMA transcripts were found to be
increased in the wild-type p53 cells but not in the p53mutant or p53-null cells (Fig. 1B). To test whether p53
activates the PUMA promoter in lung cancer cells, PUMA
reporter constructs, along with wild-type p53 and several
tumor-derived p53 mutants (10), were cotransfected into
H1299 cells. The reporter containing the p53 responsive
elements was activated by wild-type p53 but not by p53
mutants (Fig. 1C).
These results indicate that the induction of PUMA by
chemotherapeutic agents in lung cancer cells is mediated by
p53 and that this induction is abolished in p53-deficient lung
cancer cells.
PUMA profoundly suppresses growth of lung cancer cells
through induction of apoptosis. The proapoptotic function of
PUMA and lack of PUMA induction in p53-deficient lung
cancer cells prompted us to investigate whether PUMA
suppresses lung cancer cell growth. Six lung cancer cells
lines (A549, Caul 1, 128.88T, H1299, H1752, and DMS53)
were infected with an adenovirus expressing PUMA (AdPUMA) or a control adenovirus (Ad-DBH3) expressing PUMA
lacking the BH3 domain, which is essential for its proapoptotic function (14). For all six cell lines, at least 80% of the
cells were infected by Ad-PUMA and Ad-DBH3 as indicated
by the GFP signal (data not shown). After infection for 48
hours, cells were analyzed by 3-(4,5-dimethyl-thiazol-2yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
assay. Ad-PUMA was found to cause profound growth
suppression in all the cell lines whereas Ad-DBH3 had
virtually no effect on cell growth compared with the
untreated cells (Fig. 2A).
The cells infected by Ad-PUMA, but not those infected by AdDBH3, underwent massive apoptosis revealed by nuclear
staining, cell cycle analysis, and Annexin V staining (Fig. 2B
and C). Activation of caspases 3, 8, and 9 was detected in all cell
lines after Ad-PUMA infection and correlated with growth
suppression (Fig. 2A and D). Interestingly, DMS53 cells were
most sensitive to Ad-PUMA infection, showing signs of late
apoptosis 48 hours after infection, including PUMA degradation and fully activated caspases (Fig. 2A and D; ref. 14). To
examine whether PUMA-induced apoptosis in lung cancer cells
is mediated through mitochondrial pathway (26, 27), cytosolic
fractions were isolated from H1299 cells infected with AdPUMA and Ad-DBH3 for 48 hours, and then analyzed by
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Chemosensitization and Radiosensitization by PUMA
Fig. 2. PUMA suppresses the growth of lung cancer cells through induction of apoptosis. A , cell growth was measured by 3-(4,5-dimethyl-thiazol-2yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay at 48 hours following adenoviral infection (100 MOI for A549 and 128.88T; 25 MOI for others) as
described in Materials and Methods. The growth of untreated cells was defined as 100%. B, after the indicated treatments for 48 hours, H1299 cells were visualized with
phase-contrast (Phase) or fluorescence microscopy following nuclear staining with propidium iodide (PI). C, after the indicated treatments for 24 hours, H1299 cells were
stained by propidium iodide orAnnexinV/4V,6-diamidino-2-phenylindole, and then analyzed by flow cytometry to determine the fraction of sub-G1cells and early apoptotic cells
(R4), respectively. D, PUMA expression and caspase activation in lung cancer cell lines were analyzed by Western blotting 48 hours after adenoviral infection. Arrows,
active forms of caspases. E, release of cytochrome c and apoptosis-inducing factor (AIF) was examined in the cytosolic fractions of the cells following indicated treatments
by Western blotting. a-Tubulin and cytochrome oxidase subunit IV (Cox IV), which are localized in the cytosol and mitochondria, respectively, were used as controls for
fractionation.
Western blotting. Cytochrome c and apoptosis-inducing factor
were found to be released into the cytosol in the
cells infected with Ad-PUMA but not in those infected with
Ad-DBH3 (Fig. 2E).
These data show that PUMA is a potent inducer of growth
suppression and apoptosis in lung caner cells and that PUMA
promotes the release of mitochondrial apoptogenic proteins
and caspase activation to induce apoptosis in lung cancer
cells.
PUMA sensitizes lung cancer cells to chemotherapeutic agents
and g-irradiation. Abnormalities of apoptosis regulation have
been shown to contribute to the development of resistance to
chemotherapy and radiation therapy in cancer cells (4 – 6). The
important role of PUMA in DNA damage – induced and p53dependent apoptosis suggests that elevated PUMA expression
may restore sensitivity of cancer cells to anticancer agents. To
test this hypothesis, A549 lung cancer cells were treated with
low dose of Ad-PUMA [10 multiplicity of infection (MOI)],
alone or in combination with g-irradiation or chemotherapeutic agents, including Taxol, 5-FU, oxaliplatin, cisplatin, etoposide, and Adriamycin. PUMA was found to significantly
enhance the growth inhibitory effects of these chemotherapeutic drugs and g-irradiation, with the synergy most pronounced
when combined with DNA-damaging agents, including Adriamycin, cisplatin, etoposide, and g-irradiation (Fig. 3A). For
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example, as much as 8-fold increase in growth suppression was
achieved when Ad-PUMA was combined with Adriamycin. We
also determined the IC50s of several chemotherapeutic agents
in A549 cells with or without Ad-PUMA and found that
Ad-PUMA significantly lowered the IC50s of these agents by
3-fold (Adriamycin) to over 10-fold (Taxol and 5-FU; Fig. 3B;
Table 1).
We then determined whether PUMA expression sensitizes
lung cancer cells to the anticancer agents through induction of
apoptosis. A549 cells are resistant to apoptosis induced by
Adriamycin (up to 2 Ag/mL) and g-irradiation (up to 8 Gy; data
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Table 1. IC50 of the chemotherapeutics in A549 cells
with or without Ad-PUMA
Drug
Taxol
Cisplatin
5-FU
Etoposide
Adriamycin
IC50
Ad-PUMA
+Ad-PUMA
74.0 nmol/L
110.7 Amol/L
77.8 Ag/mL
48 Amol/L
0.76 Ag/mL
5.0 nmol/L
18.5 Amol/L
7.5 Ag/mL
6 Amol/L
0.22 Ag/mL
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not shown). Ad-PUMA (10 MOI) and Adriamycin (0.2 Ag/mL)
alone did not induce significant apoptosis in A549 cells
(Fig. 4A). However, almost 90% of cells underwent apoptosis
after the combination treatment for 72 hours (Fig. 4A).
Similarly, a combination of g-irradiation (8 Gy) with AdPUMA also led to a markedly enhanced apoptotic response
in A549 cells (Fig. 4B). In contrast, the control Ad-DBH3 had
no effect when combined with Adriamycin or g-irradiation
(Fig. 4A and B). Analysis of apoptosis using other approaches,
including nuclear staining and cell cycle analysis, confirmed
these results (data not shown). Furthermore, the apoptosis was
accompanied by enhanced activation of caspases 3, 8, and 9
(Fig. 4C), as well as release of cytochrome c and apoptosis-
inducing factor into the cytosol (Fig. 4E). Pretreating the cells
with caspase inhibitors significantly decreased the apoptosis
and growth inhibitory effects of the combination treatments
(Fig. 4D and data not shown). Similarly, PUMA was found to
enhance apoptosis induced by Adriamycin and g-irradiation in
128.88T cells (data not shown).
These data indicate that PUMA can synergize with different
chemotherapeutic agents and irradiation to trigger the release of
cytochrome c and apoptosis-inducing factor and caspase
activation to initiate apoptosis in lung cancer cells.
PUMA is selectively toxic to cancer cells and more potent than
p53 in growth suppression. Selectivity, or therapeutic index,
is an important issue to consider for any therapeutic agent.
Fig. 3. PUMA sensitizes lung cancer cells to chemotherapeutics and
g-irradiation. A549 cells were treated with chemotherapeutic agents
and g-irradiation, alone or in combination with Ad-PUMA orAd-DBH3
(10 MOI), as described in Materials and Methods. Cell growth
was measured by 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. A , ratios of
growth inhibition conferred by the combinations to that of drugs
alone.Taxol, 5 nmol/L; 5-FU, 25 Ag/mL; Oxa, oxaliplatin, 25 Amol/L;
Cis, cisplatin, 25 Amol/L; Etp, etoposide, 2 Amol/L; Adr, Adriamycin,
0.2 Ag/mL; IR, g-irradiation, 8 Gy. B, effects of the indicated
chemotherapeutic drugs at different concentrations, with or without
combination with Ad-PUMA, on cell growth.
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Fig. 4. The sensitizing effects of PUMA are mediated by enhanced apoptosis induction. A549 cells were subjected to the indicated treatments and analyzed for apoptosis as
described in Materials and Methods. Adenoviruses were used at 10 MOI. A and B, apoptosis was analyzed by nuclear fragmentation assays. Adriamycin, 0.2 Ag/mL;
g-irradiation, 8 Gy. C, PUMA expression and caspase activation were analyzed byWestern blotting. Arrows, active forms of caspases. D, cells were pretreated with the caspase
inhibitors (20 Amol/L) for 4 hours and then subjected to the indicated treatments for 24 or 48 hours. Apoptosis was analyzed by nuclear fragmentation assay. E, release of
cytochrome c and apoptosis-inducing factor into the cytosol was analyzed by Western blotting.
Thus, the toxicities of PUMA in several cancer and normal/
nontransformed cell lines were evaluated. Cells were transfected
with a vector expressing NH2-terminal GFP-fused wild-type
PUMA, a control vector expressing PUMA lacking the BH3
domain (DBH3), or a vector expressing the mitochondrial
localization sequence of PUMA (C43; ref. 17). Because cells
killed by PUMA do not express significant amount of GFP
(examples shown in Fig. 5A), the toxicities of PUMA to
different cell lines can be compared through analysis of GFPpositive cells after transfection. This assay is similar to the
widely used h-galactosidase cotransfection method in assessing
cytotoxicity (28). The fractions of GFP-positive cells were
indistinguishable in all cell lines after transfection with the
control vectors expressing DBH3 or C43 (Fig. 5A and B).
However, transfection with wild-type PUMA diminished GFPpositive cells in the cancer cell lines, including the lung cancer
cell lines A549, H1299, Calu 1, 128.88T, and 201T and the
colon cancer cell lines HCT116 and DLD1. In contrast, PUMA
has little effect on the normal/nontransformed cell lines,
including the primary human fibroblast cell line WI-38, the
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immortalized but nontransformed kidney epithelial cell line
293, and the retinal epithelial cell line 911 (P < 0.0001;
Fig. 5B). These results suggest that PUMA is selectively toxic to
cancer cells although the underlying mechanism of this
selectivity remains unclear.
Adenovirus-mediated transfer of p53 has been extensively
explored in cancer gene therapy (12). To test the potential
use of PUMA in cancer gene therapy, Ad-PUMA and an
adenovirus expressing p53 (Ad-p53) constructed in the same
system were compared for their potency in suppressing the
growth of lung cancer cells. Strikingly, Ad-PUMA was found
to be at least 5- to 10-fold more potent than Ad-p53 in
suppressing the growth of H1299 and A549 cells (Fig. 5C).
We observed much higher expression of PUMA but lower
expression of p21 in the cells infected with Ad-PUMA
compared with those infected with Ad-p53 at the same titer
(Fig. 5D). We also compared the chemosensitization effects
of Ad-p53 to those of Ad-PUMA and found that Ad-p53
(10 MOI) resulted in little enhancement of these effects in
A549 cells (data not shown). These results show that PUMA is
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more potent than p53 in growth suppression and chemosensitization in lung cancer cells.
PUMA suppresses tumor growth in vivo. To determine
whether PUMA confers antitumor activity in vivo, A549
xenograft tumors (f50-100 mm3) were treated with three
injections of Ad-PUMA and the control Ad-DBH3 at 5 108
plaque-forming units (Fig. 6A). To avoid potential systemic
effects of different viruses, Ad-PUMA and Ad-DBH3 were
injected into separate tumors in the same animals. Ad-DBH3
did not have any effect on tumor growth compared with PBS
alone (data not shown), with tumors reaching eight times the
initial volumes in 24 days (Fig. 6A and B). In contrast, tumors
subjected to Ad-PUMA treatment grew much slower and
reached less than twice the initial volume, with at least 80%
growth suppression compared with those treated by Ad-DBH3
(P < 0.01; Fig. 6A and B). Ad-PUMA was also used to treat
H1299 xenograft tumors and found to suppress tumor growth
by >80% (Fig. 6C). GFP fluorescence patterns indicated that
transgenes were highly expressed in the tumor cells 48 hours
following the second injection (Fig. 6D). Analyzing tumor
histology by H&E staining and apoptosis in situ by terminal
deoxyribonucleotidyl transferase – mediated dUTP nick end
labeling staining revealed significant cell loss and large fractions
of apoptotic cells in the tumors treated with Ad-PUMA but not
in those treated with Ad-DBH3 or PBS alone (Fig. 6D). These
data show that PUMA can effectively inhibit growth of
established tumors in vivo through induction of apoptosis.
Discussion
Defects in the p53 pathway, which often abolish p53mediated apoptotic response to DNA damage, are thought to
play an important role in the development of drug resistance
in cancer cells (29, 30). Although a number of proapoptotic
proteins have been identified as p53 targets, only a few have
been shown to play an important role in p53-dependent
apoptosis in gene-targeting studies (13, 31). PUMA was
identified as a p53 downstream gene that plays a critical role
in p53-dependent apoptosis. Targeted deletion of PUMA in
human colorectal cancer cells resulted in resistance to apoptosis
induced by p53, the DNA-damaging agent Adriamycin, and
hypoxia (17). Two recent studies using PUMA-knockout mice
indicated that PUMA is an essential mediator of p53-dependent
and p53-independent apoptosis in vivo (18, 19). It has also
been shown that the apoptotic mediators of p53, including
PUMA, the BH3-only proteins Bid and Noxa, and the death
receptor DR5, can be induced by chemotherapeutics and
irradiation in tissue-specific patterns, which correlated with
radiosensitivity in these tissues (32). These observations, the
prevalence of p53 mutations in lung cancer, as well as
the ineffectiveness of current lung cancer treatments, prompted
us to investigate the role of PUMA in the therapeutic responses
of lung cancer cells.
Our study showed that lung cancer cells with abnormalities in p53 are deficient in the activation of PUMA by
Fig. 5. PUMA is selectively toxic to cancer cells and more potent than p53 in growth suppression. A , A549, H1299, and 911cells were transfected with the indicated plasmids
and analyzed by fluorescence microscopy. A549 and H1299 cells are shown at 200 magnification. 911cells are shown at 400 magnification. B, the fractions of GFP-positive
cells were quantified after transfection and compared with those of GFP-C43 (100%). C, H1299 and A549 cells were infected with Ad-PUMA, Ad-p53, orAd-DBH3 at the
indicated MOI for 48 hours. Cell growth was measured by 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay.The growth
of the cells infected with Ad-DBH3 was defined as100%. D, p53, PUMA, and p21were analyzed byWestern blotting afterAd-p53 orAd-PUMA infection at the indicated MOI
for 24 hours.
Clin Cancer Res 2006;12(9) May 1, 2006
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Chemosensitization and Radiosensitization by PUMA
Fig. 6. PUMA suppresses the growth of xenograft lung tumors. A, the growth curve of A549 tumors (n = 5 per group) subjected to Ad-PUMA and Ad-DBH3 treatments
as described in Materials and Methods. Arrows, treatments were done on days 0, 2, and 4, respectively. B, top, a representative A549 tumor-bearing mouse before
(Pre-treatment) and 22 days after the indicated treatment (Post-treatment). Bottom, tumors from different treatment groups at the end of the experiment. C, growth curve of
H1299 tumors (n = 5 per group) subjected to Ad-PUMA and Ad-DBH3 treatments. Arrows, treatments were done on days 0, 3, and 6, respectively. D, frozen sections of
the H1299 tumors 48 hours following the second injection were analyzed by fluorescence microscopy to determine the efficiency of adenoviral infection (GFP, 200).
Apoptosis was analyzed by terminal deoxyribonucleotidyl transferase ^ mediated dUTP nick end labeling (TUNEL) staining (red, 200) with 4V,6-diamidino-2-phenylindole
counterstaining (blue). Tumor histology was analyzed by H&E staining (400).
chemotherapeutics and irradiation, which is likely due to
deficiency in the activation of PUMA promoter by p53. These
results suggest that lack of PUMA induction may contribute to
the development of resistance to anticancer agents in lung
cancer. Delivery of PUMA into lung cancer cells through
adenoviral gene transfer resulted in apoptosis and enhanced
sensitivity to chemotherapeutic agents and g-irradiation,
suggesting that adequate levels of PUMA are crucial for
triggering apoptotic responses to these agents. Interestingly,
PUMA seems to be the most effective in enhancing growth
suppression and apoptosis when combined with DNA-damaging agents, such as Adriamycin, etoposide, cisplatin, and girradiation (Fig. 3A). This observation is consistent with the
notion that PUMA plays a critical role in DNA damage –
induced apoptosis (17 – 19). PUMA also synergized with other
classes of chemotherapeutic agents, including the microtubule
poison Taxol and antimetabolite 5-FU, at lesser but still
significant levels (Fig. 3A). These observations suggest that
PUMA is rather a broad-spectrum chemosensitizer and radiosensitizer of lung cancer cells and warrants further evaluation.
Restoration of the p53 pathway by introducing p53 itself or
p53 downstream targets into cancer cells has become an
attractive approach in cancer gene therapy (12). Nonreplicating
adenoviruses expressing p53 have been shown to suppress cell
growth and induce apoptosis in several types of cancer in vitro
and in vivo (33, 34). Radiation therapy and p53 adenoviral
www.aacrjournals.org
gene transfer were shown to have synergistic growth-suppressive effects in a variety of cancer cells, including those of lung,
colorectal, ovarian, and head and neck cancer (35 – 38).
Clinical trials testing combinations of p53 gene replacement
with chemotherapy have yielded some encouraging results in
lung cancer patients (33, 34). Depending on its expression
level, p53 can induce cell cycle arrest or apoptosis in lung
cancer cells (39). In our study, Ad-PUMA seems to be more
potent than Ad-p53 at the same titer in growth suppression,
apoptosis induction, and chemosensitization (Fig. 5C and data
not shown). This can be explained by the much higher levels of
PUMA but lower levels of p21 induced by Ad-PUMA compared
with Ad-p53 (Fig. 5D), as high level of p21 is known to inhibit
apoptosis (17). These observations suggest that PUMA might be
a more effective radiosensitizer and chemosensitizer for lung
tumors compared with p53. However, this possibility needs to
be further evaluated in lung cancer cells and other in vivo tumor
models.
A recent study showed that PUMA can regulate diverse
apoptotic pathways by antagonizing all known antiapoptotic
Bcl-2 family members, including Bcl-2, Bcl-XL, Bcl-w, Mcl-1,
and A1, which are frequently overexpressed in cancer cells,
suggesting that PUMA may be useful for targeting a variety of
apoptotic defects in cancer cells (40). PUMA was also found
to be required for apoptosis induced by oncogenes c-myc and
E1A (18, 19). In addition, insulin-like growth factor I and
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Cancer Therapy: Preclinical
epidermal growth factor signaling pathways are frequently
deregulated in cancer and can suppress PUMA expression in
serum-starved tumor cells (16, 18, 19). Because tumor cell
growth often relies on overexpression of oncogenes or
antiapoptotic proteins (3), these observations may explain
why tumor cells are much more sensitive to PUMA than normal
cells (Fig. 5B).
Acknowledgments
We thank other members of our laboratories for helpful discussions and comments, Dr. Jill M. Siegfried at the University of Pittsburgh for providing lung cancer
cell lines, Dr. BertVogelstein at the Howard Hughes Medical Institute and the Sidney
Kimmel Comprehensive Cancer Center at Johns Hopkins for providing the p53
expression constructs, and Dr. Pamela A. Hershberger for careful reading and critical
comments.
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PUMA Sensitizes Lung Cancer Cells to Chemotherapeutic
Agents and Irradiation
Jian Yu, Wen Yue, Bin Wu, et al.
Clin Cancer Res 2006;12:2928-2936.
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