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HEMATOPOIESIS
BH3-only proteins Puma and Bim are rate-limiting for ␥-radiation– and
glucocorticoid-induced apoptosis of lymphoid cells in vivo
Miriam Erlacher, Ewa M. Michalak, Priscilla N. Kelly, Verena Labi, Harald Niederegger, Leigh Coultas, Jerry M. Adams,
Andreas Strasser, and Andreas Villunger
Numerous p53 target genes have been
implicated in DNA damage–induced apoptosis signaling, but proapoptotic Bcl-2
(B-cell leukemia 2) family members of the
BH3 (Bcl-2 homolog region [BH] 3)–only
subgroup appear to play the critical initiating role. In various types of cultured
cells, 3 BH3-only proteins, namely Puma
(p53 up-regulated modulator of apoptosis), Noxa, and Bim (Bcl-2 interacting
mediator of cell death), have been shown
to initiate p53-dependent as well as p53independent apoptosis in response to
DNA damage and treatment with antican-
cer drugs or glucocorticoids. In particular, the absence of Puma or Bim renders
thymocytes and mature lymphocytes refractory to varying degrees to death induced in vitro by growth factor withdrawal, DNA damage, or glucocorticoids.
To assess the in vivo relevance of these
findings, we subjected mice lacking Puma,
Noxa, or Bim to whole-body ␥-radiation or
the glucocorticoid dexamethasone and
compared lymphocyte survival with that
in wild-type and BCL2–transgenic mice.
Absence of Puma or Bcl-2 overexpression efficiently protected diverse types of
lymphocytes from the effects of ␥-radiation in vivo, and loss of Bim provided
lower but significant protection in most
lymphocytes, whereas Noxa deficiency
had no impact. Furthermore, both Puma
and Bim were found to contribute significantly to glucocorticoid-induced killing.
Our results thus establish that Puma and
Bim are key initiators of ␥-radiation– and
glucocorticoid-induced apoptosis in lymphoid cells in vivo. (Blood. 2005;106:
4131-4138)
© 2005 by The American Society of Hematology
Introduction
Chemotherapeutic agents and ␥-radiation can eliminate malignant
as well as normal cells by a variety of mechanisms, but increasing
evidence suggests that apoptosis plays a central role, particularly in
lymphoid cells.1,2 The apoptosis of lymphocytes in response to
genotoxic damage elicited by anticancer drugs or ␥-radiation
requires p53, the tumor suppressor inactivated in most human
malignancies.3 In response to DNA damage or activation of certain
oncogenes, wild-type (wt) p53 can either arrest cell cycle progression or induce apoptosis to prevent genome instability and cellular
transformation.4 A block to cell-cycle progression in G1 is mediated mainly by p53-regulated transcriptional activation of the
cyclin-dependent kinase inhibitor p21(WAF/CIP)5 and G2/M arrest
involves other p53 targets, such as the cytoplasmic scaffold protein
14-3-3␴ and the proliferating cell nuclear antigen (PCNA)–binding
protein termed growth arrest and DNA damage protein 45
(GADD45).6-8 More than 20 genes have been implicated in
p53-mediated apoptosis,3 and p53 has also been proposed to have a
direct role in cell death initiation at the mitochondrial membrane.9,10
Among the candidate proapoptotic p53 target genes, members
of the BH3 (Bcl-2 homology region)–only subgroup of proapoptotic Bcl-2 (B-cell leukemia 2)–like molecules stand out, since
these proteins have been recognized as essential initiators of
programmed cell death in species as distantly related as Caenorhab-
ditis elegans (C elegans) and mice.11 The BH3-only proteins, such
as Bim (Bcl-2 interacting mediator of cell death), Puma (p53
up-regulated modulator of apoptosis), and Noxa, can be activated
transcriptionally or after translation in response to developmental
cues and cytotoxic stimuli, including anticancer drugs.12 Their
proapoptotic activity relies on their ability to bind and antagonize
prosurvival members of the Bcl-2 family,13 but the proapoptotic
Bcl-2 family members Bax and Bak14 are required for apoptosis
induced by BH3-only proteins.15 The Bax (Bcl-2 associated X
protein) Bak (Bcl-2 homologous killer)–like proteins are thought to
permeabilize the outer mitochondrial membrane, causing release of
apoptogenic molecules, such as cytochrome c or Smac/Diablo, that
promote activation of the caspases that demolish the cell.15
Resistance to anticancer therapy, particularly in hematologic
malignancies, has been associated with overexpression of Bcl-2 or
its prosurvival homologs, inactivation of Bax, or loss of p53
function.2 p53 drives the transcription of the BH3-only protein
genes Puma16-18 and Noxa,19 and cultured lymphocytes from
Puma-deficient mice were shown to be refractory to apoptosis
induced by ␥-radiation and certain chemotherapeutic drugs.20,21
Noxa appears to play a more restricted role, as it proved dispensable for p53-mediated apoptosis in lymphocytes in vitro but is
required for apoptosis in oncogene-transformed mouse embryonic
From the Division of Experimental Pathophysiology & Immunology, Biocenter,
Innsbruck Medical University, Innsbruck, Austria; and The Walter and Eliza Hall
Institute of Medical Research, Melbourne, Australia.
a DOC-FFORTE Fellow of the Austrian Academy of Science (OEAW).
Submitted April 20, 2005; accepted August 11, 2005. Prepublished online as
Blood First Edition Paper, August 23, 2005; DOI 10.1182/blood-2005-04-1595.
Supported by grants from the National Health and Medical Research Council
(NHMRC) (Canberra), the Dr Josef Steiner Cancer Research Foundation
(Bern), the Leukemia and Lymphoma Society, the National Institutes of Health
(NIH) (A.S. and J.M.A.), and the Austrian Science Fund (FWF) Projects R15,
START, and SFB021 (“Cell proliferation and cell death in tumors”) (A.V.). V.L. is
BLOOD, 15 DECEMBER 2005 䡠 VOLUME 106, NUMBER 13
The online version of the article contains a data supplement.
Reprints: Andreas Villunger, Division of Experimental Pathophysiology
Biocenter, Innsbruck Medical University, A-6020 Innsbruck, Austria; e-mail:
[email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2005 by The American Society of Hematology
4131
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BLOOD, 15 DECEMBER 2005 䡠 VOLUME 106, NUMBER 13
ERLACHER et al
fibroblasts, in which Puma also plays a critical role.21,22 Puma
deficiency also protected lymphocytes against certain p53independent cell death stimuli, such as cytokine deprivation or
treatment with glucocorticoids (GCs).20,21 Similarly, Bim is essential for lymphocyte apoptosis induced by cytokine withdrawal or
calcium flux, and it was proposed to play a minor role in cell death
induced by ␥-radiation or treatment with GCs.23 Within the animal,
Bim is required for negative selection of autoreactive T cells24-26 as
well as B cells27 and for termination of T-cell immune responses,28,29 therefore precluding autoimmunity.
In order to evaluate the physiologic relevance of these in vitro
findings for the effects of anticancer therapy in vivo, we examined
the responses of mice lacking the BH3-only proteins Puma, Noxa,
or Bim to whole-body ␥-radiation or the glucocorticoid dexamethasone. The effects on lymphocyte survival were compared with
those observed in wild-type mice and animals expressing a
vav-BCL2 transgene, which is highly expressed in all lymphoid
populations.30 We show that absence of Puma or Bcl-2 overexpression efficiently protects lymphocytes from the effects of DNA
damage in vivo and that loss of Bim affords limited protection,
mainly in the T-cell linage, whereas absence of Noxa has no
protective effect. Moreover, we show that absence of either Bim or
Puma provides cell type–specific protection from the effects
of GCs.
anti-CD8; RB6-8C5, anti–Gr-1; S7, anti-CD43; 5.1, anti–immunoglobulin
M (IgM); 11/26C, anti-IgD; MI/70, anti–Mac-1; Ter119, anti–erythroid cell
surface marker; and T24.31.2, anti–Thy-1.
Materials and methods
Results
Mice
Loss of Puma or Bim protects thymocytes from apoptosis
induced by whole-body ␥-radiation
All animal experiments were performed in accordance with the Austrian
“Tierversuchsgesetz” and have been granted by the Bundesministerium für
Bildung, Wissenschaft und Kultur or were performed according to the
guidelines of the Melbourne Directorate Animal Ethics Committee. The
generation and genotyping of the Puma-deficient, Noxa-deficient,21 Bimdeficient,23 and vav-BCL2–transgenic mice30 have been described previously. p53⫺/⫺ mice were kindly donated by Manuel Serrano (Centro
Nacional de Investigaciones Oncológicas, Madrid). All mouse strains were
on an inbred C57BL/6 genetic background or in the case of the Bim⫺/⫺ mice
had been backcrossed onto this background for at least 12 generations.
Radiation experiments
Adult (7-10 weeks old) mice of the relevant genotypes were exposed to 2.5
or 5.0 Gy ␥-radiation in a linear accelerator. Mice were killed by cervical
dislocation 20 hours after treatment. Total numbers of cells in bone marrow,
thymus, spleen, lymph nodes, and peripheral blood were determined by
preparing single-cell suspensions from these tissues and counting an aliquot
stained with trypan blue in a hemocytometer.
Glucocorticoid treatment of mice
Adult (7-10 weeks old) mice were injected intraperitoneally with graded
doses of the GC dexamethasone, dissolved in normal saline containing 20
␮g/mL propyleneglycol. After 20 hours, mice were killed by cervical
dislocation.
Immunofluorescence staining, flow cytometric analysis,
and cell sorting
Single-cell suspensions from bone marrow, lymph nodes, spleen, and
thymus were surface stained with monoclonal antibodies conjugated with
fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin
(APC), or biotin (Molecular Probes, Eugene, OR). The monoclonal
antibodies used and their specificities are as follows: RA3-6B2, anti-B220;
GK1.5, anti-CD4; H129.19.6.8, anti-CD4; 53.6.72, anti-CD8; YTS 169,
TUNEL staining of tissue sections
To identify apoptotic cells in tissues, sections were freed of paraffin,
dehydrated, and incubated for 60 minutes in a humidified incubator with
terminal deoxynucleotidyl transferase in the presence of FITC–deoxyuridine triphosphate (dUTP) using an in situ cell death detection kit according
to the manufacturer’s recommendation (ROCHE, Milan, Italy). Sections
were counterstained using 7-aminoactinomycin D (7-AAD; Sigma, St
Louis, MO), and incorporated FITC-dUTP terminal deoxynucleotidyl
transferase–mediated dUTP nick end labeling–positive (TUNEL⫹) cells
within sections were evaluated using a fluorescence microscope (Zeiss
Axiovert 100 M; Carl Zeiss, Heidelberg, Germany) equipped with Plan
Neofluar 20 ⫻/0.5 NA and 40 ⫻/0.75 NA objective lenses (Zeiss). Sections
were embedded in Mowiol/DABCO. Images were acquired using LSM
510, version 2.8 SP1 (Carl Zeiss).
Statistical analysis
Statistical analysis was performed using the Student t test and Stat-view 4.1
software (SAS Institute, Cary, NC). P values of less than .05 were
considered to indicate statistically significant differences.
To analyze the requirement of individual BH3-only proteins for
DNA damage–induced apoptosis of lymphoid cells in vivo, we
exposed wild-type animals and mice lacking Puma, Noxa, or Bim,
as well as animals expressing a BCL2 transgene in all hematopoietic cell types,30 to graded doses of ␥-radiation. Animals were
killed 20 hours thereafter, and thymic glands were harvested and
analyzed for their cellularity and cell subset composition as well as
for apoptosis in situ. Immature CD4⫹8⫹ double-positive (DP)
thymocytes are highly susceptible to the effects of ␥-radiation,31
and flow cytometric analysis using cell surface marker–specific
antibodies demonstrated a dose-dependent decrease in the percentages of these cells in wt and Noxa-deficient animals (Figure 1A,C).
In contrast, the percentages of DP thymocytes lacking Puma and
Bim or those overexpressing Bcl-2 dropped only marginally
(Figure 1A,C). Determination of total thymocyte numbers (Figure
1B) and absolute numbers of CD4⫹8⫹ DP cells (Figure 1D)
confirmed that Puma-deficient and BCL2–transgenic thymocytes
were highly protected from the effects of ␥-radiation in vivo and
that Bim⫺/⫺ cells displayed lower, yet significant, resistance,
whereas Noxa⫺/⫺ DP thymocytes were killed as efficiently as wt
cells (Figure 1B-D).
In order to investigate ␥-radiation–induced apoptosis in the
thymus in situ, we performed TUNEL staining of nicked DNA on
thymic sections of wt, Puma-deficient, and Bim-deficient animals.
While wt thymic glands had lost their normal architecture within 20
hours after exposure to ␥-radiation, those from Puma-deficient
mice maintained clearly demarcated cortical and medullary structures (compare Figure 2A-C with 2D-F). Sections from untreated
mice of all genotypes showed relatively few TUNEL⫹ (green)
apoptotic cells (Figure 2A,D,G), but numerous green cells became
apparent at higher magnification in thymic sections of irradiated wt
mice (Figure 2C). In contrast, few green cells appeared in sections
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BLOOD, 15 DECEMBER 2005 䡠 VOLUME 106, NUMBER 13
Puma AND Bim MEDIATE ANTICANCER THERAPY EFFECTS
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Figure 1. Puma and Bim are rate limiting for ␥-radiation–induced apoptosis of thymocytes in vivo. Animals of the indicated genotypes were exposed to the
indicated doses of whole-body ␥-radiation and killed 20
hours thereafter. Thymi were harvested and the singlecell suspensions stained with fluorescence-conjugated
antibodies to CD4 and CD8 and analyzed by flow
cytometry. (A) Representative dot blots of stained thymocyte suspensions from untreated or radiated animals of
each genotype indicating the percentage of CD4⫺8⫺,
CD4⫹8⫹, CD4⫹8⫺, and CD4⫺8⫹ cells. The lower proportion of CD4⫹8⫹ DP cells in the thymus of untreated mice
that lack Bim or overexpress Bcl-2 is expected from
previous studies.23,30 (B) Thymic cellularity of untreated
or radiated animals was assessed using a hemocytometer and trypan blue staining. (C) The percentage of
CD4⫹8⫹ DP cells and total thymic cellularity were used to
calculate (D) the absolute number of CD4⫹8⫹ DP thymocytes in control and radiated animals. Bars represent
means ⫾ SE of 4 to 10 animals of each genotype and
treatment regimen from at least 4 independent experiments. tg indicates transgenic. Statistically significant
differences are as follows: (D) 2.5 Gy wt vs Puma⫺/⫺
(P ⬍ .001), 5.0 Gy wt vs Puma⫺/⫺ (P ⬍ .001), 2.5 Gy wt
vs Bim⫺/⫺ (P ⫽ .003), 5.0 Gy wt vs Bim⫺/⫺ (P ⫽ .001),
2.5 Gy wt vs vav-BCL2 (P ⬍ .001), 5.0 Gy wt vs vavBCL2 (P ⬍ .001).
Figure 2. ␥-Radiation–induced apoptosis of thymocytes is mediated by a Puma- and Bim-dependent
mechanism. Thymic sections derived from wt (A-C),
Puma-deficient (D-F), and Bim-deficient (G-I) animals 20
hours after exposure to 2.5 Gy ␥-radiation were TUNEL
stained using FITC-dUTP to detect nicked DNA in apoptotic cells. Nuclei were counterstained using 7-AAD. Sections were analyzed using a ZEISS Axiovert fluorescence
microscope. The top panels show stains of representative thymic sections from untreated animals, revealing
comparable numbers of TUNEL⫹ (green) cells in all
genotypes analyzed. After exposure to 2.5 Gy ␥-radiation, marginal zone and cortical structures are no longer
visible in the wt (B) and are reduced in Bim⫺/⫺ (H) thymic
sections but clearly maintained in the Puma⫺/⫺ thymus
(E). At higher magnification (bottom panels), sections
from wt mice (C) exhibit significantly more TUNEL⫹
apoptotic cells than those from Puma⫺/⫺ or Bim⫺/⫺
animals (F,I). The images are representative of 3 or more
independent stains performed on organs of at least 2
animals per genotype and treatment.
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ERLACHER et al
BLOOD, 15 DECEMBER 2005 䡠 VOLUME 106, NUMBER 13
Figure 3. Puma mediates ␥-radiation–induced apoptosis of mature T and B lymphocytes and B-cell precursors in vivo. Animals of the indicated genotypes were left
untreated or exposed to the indicated doses of whole-body ␥-radiation and killed 20 hours thereafter. Spleen and bone marrow were harvested, and the single-cell suspensions
were counted and stained with fluorescence-conjugated antibodies specific for CD4 and CD8 to identify mature T cells or IgM and IgD to identify mature B cells using a flow
cytometer. Bone marrow suspensions were stained using antibodies recognizing B220, IgM, and CD43 in order to identify pre-B-cell precursors. The total cellularity of CD4⫹ T
cells, CD8⫹ T cells, IgM⫹D⫹ B cells in spleen (A-C), and B220⫹sIgM⫹CD43⫺ pre-B cells found in the bone marrow of both femora (D) is depicted. Bars represent means ⫾ SE
of 4 to 10 animals of each genotype and treatment regimen used in at least 4 independent experiments. Statistically significant differences are as follows: (A) CD4⫹ T cells: wt
versus Puma⫺/⫺ (P ⬍ .001), wt versus Bim⫺/⫺ (P ⬍ .012), wt versus vav-BCL2 (P ⬍ .001); (B) CD8⫹ T cells: wt versus Puma⫺/⫺ (P ⬍ .007), wt versus Bim⫺/⫺ (P ⬍ .013), wt
versus vav-BCL2 (P ⬍ .002); (C) IgM⫹D⫹ B cells: wt versus Puma⫺/⫺ (P ⬍ .001), wt versus Bim⫺/⫺ (P ⬍ .029), wt versus vav-BCL2 (P ⬍ .001); (D) B220⫹sIgM⫺CD43⫺ pre B
cells: wt versus Puma⫺/⫺ (P ⬍ .001), wt versus vav-BCL2 (P ⬍ .002).
from Puma-deficient animals (Figure 2E-F). In sections from
Bim⫺/⫺ mice, although cortical regions were strongly reduced after
exposure to ␥-radiation (Figure 2H-I), there were fewer TUNEL⫹
cells than in sections from wt mice (compare Figure 2C with 2I).
Taken together, these experiments demonstrate that in developing T lymphocytes the rate-limiting BH3-only protein for p53mediated apoptosis in response to ␥-radiation in vivo is Puma, but
that Bim also plays a minor role, whereas Noxa is dispensable.
Mature T and B lymphocytes and pre-B cells require Puma
for DNA damage–induced apoptosis in vivo
The sensitivity to ␥-radiation as well as the molecular requirements
for DNA damage–induced apoptosis have been reported to differ
between immature thymocytes and mature lymphocytes.1,32 Therefore, we investigated the impact of whole-body ␥-radiation on the
cellular composition of peripheral lymphoid organs, including
spleen, lymph nodes, and bone marrow, as well as peripheral blood.
Again, wt, Puma⫺/⫺, Noxa⫺/⫺, Bim⫺/⫺, and vav-BCL2–transgenic
mice were killed 20 hours after exposure to graded doses of
␥-radiation. Our analysis revealed that loss of Puma or Bcl-2
overexpression conferred significant protection against ␥-radiation
on all the lymphoid populations examined: CD4⫹8⫺ and CD4⫺8⫹
peripheral T cells (Figure 3A-B), naive IgM⫹IgDlo B cells (data not
shown), mature IgM⫹IgD⫹ B cells from spleen (Figure 3C) and
lymph nodes (not shown), lymphocytes from peripheral blood
(Figure S1, available on the Blood website; see the Supplemental
Figures link at the top of the online article), and immature
B220⫹sIgM⫺CD43⫺ pre-B cells from bone marrow (Figure 3D).
Consistent with our previous observations with thymocytes (Figure
1), absence of Bim also provided some protection to mature T and
B cells (Figure 3A-C). For example, 20 hours after 2.5 Gy radiation
a mean of 28.08% ⫾ 0.5% of wt CD4⫹ T cells and 20.5% ⫾ 0.2%
of wt CD8⫹ T cells remained, when compared with untreated controls,
whereas 40.14% ⫾ 1.5% of Bim⫺/⫺ CD4⫹ and 34.61% ⫾ 0.9% of
Bim⫺/⫺ CD8⫹ T cells survived this treatment (Figure 3A-B). Bim
deficiency did not, however, protect the pre-B cells (Figure 3D) and
Noxa deficiency had no protective effect in any of the lymphoid
populations (Figure 3).
TUNEL staining of untreated spleens failed to reveal apoptotic cells in situ (Figure 4A-C), but the spleens from ␥-irradiated
wt animals exhibited many apoptotic TUNEL⫹ green cells
(Figure 4B). Consistent with our flow cytometric analysis
(Figure 3), sections from irradiated Puma- or Bim-deficient
animals reproducibly displayed far fewer TUNEL⫹ cells than wt
sections (Figure 4D-F).
Taken together, these observations indicate that ␥-radiation–
induced apoptosis of mature T and B cells in vivo depends mainly
on Puma but that Bim also contributes significantly. In pre-B cells,
Figure 4. In situ analysis of ␥-radiation–induced
apoptosis of splenocytes. Sections from untreated (top
panels) or ␥-radiated (bottom panels) spleens derived
from wt (A-B), Puma-deficient (C-D), and Bim-deficient
(E-F) animals 20 hours after exposure to 5.0 Gy were
TUNEL stained using FITC-dUTP to detect nicked DNA in
apoptotic cells. Nuclei were counterstained using 7-AAD.
Sections were analyzed using a ZEISS Axiovert fluorescence microscope. Analysis of sections failed to reveal
TUNEL⫹ apoptotic cells in control animals (A,C,E). Numerous TUNEL⫹ apoptotic cells are visible in sections derived from wt animals (B), but significantly fewer green
cells appear in Puma⫺/⫺ or Bim⫺/⫺ sections (C,E). Representative images of 3 or more independent stains performed on organs of at least 2 animals per genotype and
treatment are shown.
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␥-radiation–induced death depends primarily on Puma and presumably on other unidentified cell death mediators.
Both Puma and Bim are required for dexamethasone-induced
apoptosis of lymphoid cells
GCs are widely used in anticancer therapy and are proving
particularly effective in the treatment of certain childhood leukemias.33 Their proapoptotic effects on normal lymphocytes are well
documented, but the molecular basis is still poorly understood.
Bcl-2 overexpression confers resistance to GCs upon normal
thymocytes, pre-B cells, mature T as well as B cells, and
lymphoma-derived cell lines,31,34 and GC transcriptional targets
include the BH3-only genes Bim and Puma, making them likely
candidates for initiating this apoptotic pathway.18,33,35-37 In order to
evaluate the in vivo contribution of BH3-only proteins to GCinduced apoptosis of lymphocytes, wt, Puma⫺/⫺, Bim⫺/⫺, and
vav-BCL2–transgenic animals were injected intraperitoneally with
graded doses of the therapeutically used glucocorticoid dexamethasone. Animals were killed 20 hours thereafter, and thymic glands,
spleens, and bone marrow samples were analyzed for their
cellularity and cell subset composition.
Total thymic cellularity dropped drastically in wt but not in
vav-BCL2–transgenic animals (Figure 5A). Total cellularity in
Figure 6. Cell type–specific contributions of Puma and Bim to glucocorticoidinduced apoptosis of immature and mature lymphocytes in vivo. Animals of the
indicated genotypes were injected intraperitoneally with graded doses of dexamethasone and killed 20 hours later. Spleens and bone marrow were harvested, and
single-cell suspensions were counted and stained with various fluorescenceconjugated cell surface marker–specific antibodies and analyzed in a flow cytometer.
Total numbers of mature CD4⫹ T cells (A) and CD8⫹ T cells (B) from spleens of saline
or dexamethasone-injected animals. (C) Total cellularity of B220⫹sIgM⫺CD43⫺ pre-B
cells in both femora of untreated or dexamethasone-treated animals. Bars represent
means ⫾ SE of 3 to 6 animals of each genotype and treatment regimen used from at
least 3 independent experiments. Statistically significant differences are as follows:
(A) CD4⫹ T cells: wt versus Puma⫺/⫺ (P ⬍ .049); wt versus vav-BCL2 (P ⬍ .008); (B)
CD8⫹ T cells: wt versus Puma⫺/⫺ (P ⬍ .035), wt versus vav-BCL2 (P ⬍ .007); (C)
B220⫹IgM⫺CD43⫺ pre B cells: wt versus Bim⫺/⫺ (P ⬍ .022).
Figure 5. Delay of glucocorticoid-induced apoptosis in Puma- or Bim-deficient
thymocytes. Animals of the indicated genotypes were injected intraperitoneally with
graded doses of dexamethasone and killed 20 hours later. Thymi were harvested,
and single-cell suspensions were counted and stained with fluorescence-conjugated
antibodies to CD4 and CD8 and analyzed in a flow cytometer. (A) Total thymic
cellularity of saline or dexamethasone-injected animals. (B) The percentage of
CD4⫹8⫹ double-positive cells and total thymic cellularity were used to calculate (C)
the absolute number of immature CD4⫹8⫹ DP thymocytes present in control and
dexamethasone-treated animals. Bars represent means ⫾ SE of 3 to 6 animals of
each genotype and treatment regimen from at least 3 independent experiments.
Statistically significant differences are as follows: (C) CD4⫹8⫹ DP thymocytes: wt
versus Puma⫺/⫺ (P ⬍ .033), wt versus Bim⫺/⫺ (P ⬍ .05), wt versus vav-BCL2
(P ⬍ .013). Thymic sections derived from wt (D), Puma-deficient (E), and Bimdeficient (F) animals 20 hours after exposure to 250 ␮g dexamethasone were TUNEL
stained using FITC-dUTP to detect nicked DNA in apoptotic cells. Nuclei were
counterstained using 7-AAD. Sections were analyzed using a ZEISS Axiovert
fluorescence microscope. The images are representative of 2 independent stains
performed on organs of at least 2 animals per genotype and treatment.
Puma- and Bim-deficient thymi was also strongly reduced, but not
as pronounced as in thymi from wt animals (Figure 5A). CD4⫹8⫹
DP immature thymocytes are particularly sensitive to the effects of
GCs, responding with rapid apoptosis.31,34 Immunofluorescent
staining and flow cytometric analysis demonstrated a substantial
decrease in the percentages of DP thymocytes in dexamethasoneinjected wt animals, but not in mice lacking Puma or Bim, or those
overexpressing Bcl-2 (Figure 5B). Calculating absolute numbers of
CD4⫹8⫹ DP cells demonstrated that Puma-deficient as well as
Bim-deficient thymocytes were partially protected from the effects
of GCs (Figure 5C), and, as shown before,31,34 Bcl-2 overexpression potently protected CD4⫹8⫹ DP thymocytes from dexamethasone-induced killing (Figure 5C). Consistent with a minor protective effect of Puma or Bim deficiency, a significant number of
TUNEL⫹ green apoptotic cells was detected, predominantly in
cortical regions of thymic sections from wt, Puma-deficient, and
Bim-deficient animals (Figure 5D-F and data not shown).
Since mature lymphocytes are sensitive to GCs, albeit much
less so than DP thymocytes, we also evaluated the impact of
dexamethasone on the survival of mature splenic lymphocytes.
While both Puma and Bim contributed to thymocyte apoptosis,
selectivity became apparent in the periphery. Puma deficiency
reduced the deletion of both CD4⫹ and CD8⫹ mature T cells from
spleen in response to dexamethasone, but absence of Bim provided
no statistically significant degree of protection (Figure 6A-B).
Mature wt B-cell numbers also fell about 50% in response to
systemic GC treatment, but neither absence of Puma or Bim nor
Bcl-2 overexpression prevented this drop (data not shown). In
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4136
ERLACHER et al
marked contrast to the T cells, absence of Bim potently protected
immature pre-B cells from the bone marrow, whereas absence of
Puma had no protective effect (Figure 6C). Collectively, our data
indicate that both Puma and Bim play critical and, depending on the
cell type, partially overlapping roles in GC-induced apoptosis of
lymphocytes in vivo.
Discussion
The resistance of malignant cells to ␥-radiation or anticancer drugs,
a widespread problem during anticancer therapy, is often attributable to elevated levels of prosurvival Bcl-2 family members,
inactivation of proapoptotic Bcl-2 family members, or loss of the
tumor suppressor p53.1,2 In addition, killing of normal lymphoid
and myeloid cells can be a serious unwanted side effect of
anticancer therapy. We have used a panel of knock-out and
transgenic animals to investigate the in vivo role of the 3 BH3-only
proteins, Puma, Noxa, and Bim, in lymphocyte apoptosis induced
by ␥-radiation and GCs. Quantitative analysis by flow cytometry
(Figures 1 and 3) and in situ analysis of the thymus (Figure 2) and
spleen (Figure 4) revealed that in the presence of functional p53,
Puma is the rate-limiting proapoptotic molecule for ␥-radiation–
induced apoptosis in diverse lymphoid cells, including thymocytes,
mature T and B cells, and pre-B cells (Figures 1-4). Indeed, the
protection conveyed by its loss in thymocytes (Figures 1-2) and
CD4⫹ and CD8⫹ mature T cells and B cells (Figure 3) approached
that given by the highly expressed vav-BCL2 transgene.
Somewhat surprisingly, both the flow cytometric and the in situ
TUNEL analysis also indicated that Bim, which is not a primary
target of p53, contributes to ␥-radiation–induced death in thymocytes and mature lymphocytes (Figure 1-4). These data are
consistent with previous in vitro observations revealing a small but
significant survival advantage for Bim⫺/⫺ thymocytes23 and granulocytes38 in response to DNA damage caused by ␥-radiation or
etoposide, respectively. These findings may indicate that genotoxic
damage also elicits a p53-independent route to cell death that
requires Bim. It is possible that p53-induced genes either influence
Bim mRNA expression levels or regulate its protein function after
translation by mediating its activation. Alternatively, since both
Puma and Bim can bind to the same set of prosurvival Bcl-2
homologs with similar affinities,48 the absence of Bim might
simply increase the availability of free prosurvival Bcl-2–like
molecules now able to antagonize Puma more effectively. At first
sight, the latter model seems unlikely, because it would predict, for
example, that loss of Bim would increase the resistance of
thymocytes to apoptosis induced by phorbol ester, which requires
Puma, and that loss of Puma should render thymocytes resistant to
cell death induced by Ca⫹⫹ flux, which requires Bim, but neither of
these is the case (Villunger et al21 and M.E. and A.V., unpublished
observations, 2005). However, the visibility of such an indirect
beneficial effect may also depend on the pattern of prosurvival
homologs expressed in a given cell type.
Bcl-2 overexpression proved more potent than loss of Puma in
protecting CD4⫹8⫹ DP thymocytes (Figure 1D) and pre-B cells
(Figure 3D) from ␥-radiation. This might be interpreted that these
cells can die in a p53-independent but Bcl-2–blockable manner.
That appears unlikely, however, because DNA damage–induced
apoptosis independent of p53 has so far been observed only in
cycling normal lymphoid or lymphoma cells,32 while most CD4⫹8⫹
DP thymocytes and pre-B cells are noncycling and are rendered
completely resistant to ␥-radiation by loss of p53.32,39,40
BLOOD, 15 DECEMBER 2005 䡠 VOLUME 106, NUMBER 13
At present, we cannot exclude that ␥-radiation–induced cell
cycle arrest in surviving ␤-selected CD4⫺8⫺ double-negative (DN)
cells contributes to the net loss of DP thymocytes. However, the
limited number of DN cells that dies in response to ␥-radiation
within 20 hours does so in a strictly p53-dependent, Bcl-2–
blockable manner (Figure S2).
Activation of death receptor signaling by ␥-radiation–induced
activation of p53 also appears unlikely to play a role in these cells,
because this death pathway is not inhibited by Bcl-2 overexpression,41,42 and loss of functional FADD or Casp-8, which are
essential for all death receptor–mediated killing, does not interfere
with DNA-damage–induced apoptosis.41,43,44 The explanation
we favor is that p53, directly or indirectly, activates additional
BH3-only proteins that can cooperate with Puma in ␥-radiation–
induced apoptosis of thymocytes and pre-B cells in vivo,
although a contribution by other cell death mediators cannot
currently be excluded.
The available data from gene knock-out mice, however, do not
seem to support a role for several BH3-only candidates: no
protection of lymphocytes from the effects of ␥-radiation, in vivo
or in vitro, has been observed on loss of Noxa, a bona fide
transcriptional target of p53 (this study and Shibue et al22) nor of
Bid,45 Bik,46 or Bad (A.S., unpublished data, 2005). As the most
likely candidate, this leaves Bim, which has been suggested to act
in parallel to p53 in preventing lymphomagenesis.47 Nevertheless,
even BH3-only proteins that on their own do not contribute notably
to ␥-radiation–induced apoptosis might cooperate with Puma in
this process, and generation of mice lacking Puma plus another
BH3-only protein (eg, Puma plus Noxa or Puma plus Bim) should
clarify this issue. At present, however, the possibility, suggested by
other groups,9,10 that p53 has a transcription-independent role in
initiating cell death involving its direct action at mitochondria
cannot be excluded.
Of interest, Bim-deficient thymocytes of all maturation stages
(CD4⫺8⫺, CD4⫹8⫹, CD4⫹8⫺, and CD4⫺8⫹) appeared to be
equally sensitive to the effects of ␥-radiation (Figure 1) or GCs
(Figure 5) and died with similar kinetics, whereas in wt animals, as
observed before,31 CD4⫹8⫹ DP thymocytes are the most sensitive
thymocyte subset. This observation explains why the high percentages of CD4⫹8⫹ DP cells remaining after radiation (or GC
injection) of Bim⫺/⫺ mice did not lead to a greater increase in the
number of surviving CD4⫹8⫹ DP cells (compare Figure 1C vs 1D
and 5B vs 5C). The molecular basis for this finding is currently
under investigation.
Although Noxa mRNA levels were reported to increase in
thymocytes after ␥-radiation (Oda et al19; and L.C. and A.S.,
unpublished data, 2005), Noxa-deficient lymphocytes of all differentiation stages proved normally sensitive to this death stimulus.
This is somewhat surprising, since transcriptional activation of
Noxa clearly requires p53.19,22 A plausible explanation arises from
the recent finding that Noxa effectively engages only the prosurvival Bcl-2–like molecules Mcl-1 (myeloid cell leukemia 1) and
A1 but not Bcl-2 or Bcl-XL.48 Since lymphocyte survival depends
mainly on combined action of Bcl-2, Bcl-XL, and Mcl-1,49 the
inability of Noxa to engage Bcl-2 and Bcl-XL would explain why
its apoptotic role in lymphocytes is far less pronounced than that of
Puma and Bim, which engage all the prosurvival family members.48 The generation of compound knock-out mice lacking Puma
or Bim as well as Noxa will establish whether Noxa contributes
significantly to p53-mediated apoptosis in lymphocytes.
Taken together, our in vivo findings correlate surprisingly well
with studies performed on isolated lymphocytes exposed to ␥-radiation ex vivo,20-23 indicating that the microenvironment does not
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
BLOOD, 15 DECEMBER 2005 䡠 VOLUME 106, NUMBER 13
play a critical role in the regulation of the DNA-damage response
in lymphocytes in vivo. The only indication that the microenvironment may induce different cellular responses can be drawn from
our observation that loss of Puma or Bcl-2 overexpression provides
only limited protection to peripheral blood lymphocytes (Figure
S2). However, this observation could instead mean that damage to
the endothelium allows extravasation of lymphocytes into the
surrounding tissue and that this endothelial damage is not
blocked efficiently by absence of p53 or Puma. This is supported
by the fact that peripheral blood lymphocytes also disappeared
readily from the blood stream of Puma- and, most importantly,
p53-deficient animals, but these same cell types exhibit resistance to ␥-radiation–induced death in the spleen (Figure 3,
Figure S2, and data not shown).
GCs are an essential component of many protocols for treatment of lymphoid malignancies.33 Childhood T-cell leukemias, for
example, are highly sensitive to GCs, and primary sensitivity to
GC-induced apoptosis is associated with good prognosis.33 In
contrast, primary or treatment-induced resistance to GCs, mostly
associated with mutations in the GC receptor (GR), generally
indicates an unfavorable course of the disease.36 GC treatment
leads to transcriptional activation or repression of numerous target
genes, but none was known that induces apoptosis directly until the
genes for both Bim and Puma were shown to be targets.18,35,37
Our in vivo analysis of GC sensitivity of lymphoid cells in
BH3-only knock-out mice agrees with observations from cultured
cells.21,23 Absence of Puma or Bim provided significant protection
from GC-induced apoptosis in thymocytes, but overexpressed
Bcl-2 was more effective (Figure 5). This indicates that Bim and
Puma act in an overlapping manner, and experiments to determine
whether Bim/Puma double-deficient thymocytes are completely
resistant to GCs have been initiated.
The relative significance of Puma and Bim in GC-induced death
seems to vary with cell type. Whereas their role in thymocytes
appears comparable (Figure 5A) in mature CD4⫹ and CD8⫹ T
cells, which are less sensitive to GCs than CD4⫹8⫹ DP thymocytes,
Puma loss conveyed greater protection than Bim loss and indeed
was at least as effective as Bcl-2 overexpression (Figure 6A-B).
In striking contrast, in pre-B cells, loss of Bim provided almost
complete resistance to GC treatment, whereas loss of Puma had no
Puma AND Bim MEDIATE ANTICANCER THERAPY EFFECTS
4137
protective effect (Figure 6C). These results are in line with
observations that Bim is strongly induced by dexamethasone
treatment in the GC-sensitive pre-B-cell leukemia cell line 697.35,36
It thus appears that Puma and Bim can be induced and/or activated
by GCs in a cell type–specific manner.
Collectively, our experiments demonstrate that Puma is the
rate-limiting BH3-only protein mediating p53-induced apoptosis in
response to ␥-radiation in vivo, whereas Bim plays a minor role and
Noxa is dispensable, at least in lymphoid cells. Indeed, except in
DP thymocytes, Puma appears responsible for almost all the
proapoptotic effects of p53 in the lymphoid compartment. In
GC-induced apoptosis of lymphocytes, however, both Puma and
Bim have major roles (Figures 5-6). From the therapeutic perspective, it is noteworthy that both BH3-only proteins can be upregulated by GCs and other mechanisms that do not require
p53.12,18 Hence, exploring their regulation in more detail may well
prompt novel strategies for rendering tumors with defective p53
status, or those refractory to glucocorticoid treatment (eg, due to
GR mutations), more sensitive to conventional anticancer treatment. Furthermore, it appears likely that the propensity of malignant lymphocytes to induce these genes in response to ␥-radiation
or GCs will significantly influence treatment outcome. Hence, the
ability of tumors to induce Puma and/or Bim expression under
different treatment regiments may well provide useful prognostic
markers to guide antitumor therapy.
Acknowledgments
We are grateful to F. Müllauer, K. Rossi, and C. Manzl for technical
assistance and mouse genotyping; to M. Brennsteiner for animal
care; to Prof P. Lukas for enabling radiation experiments; to R.
Pfeilschifter for histology sections; and to R. Gruber-Sgonc for
help with TUNEL analysis. We thank Profs S. Cory, R. Kofler, G.
Wick, S. Kiessling, and all our colleagues in the lab for insightful
discussions.
Dr Coultas’s current address is Samuel Lunenfeld Research
Institute, Rm 884, Mount Sinai Hospital, University of Toronto,
600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
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2005 106: 4131-4138
doi:10.1182/blood-2005-04-1595 originally published online
August 23, 2005
BH3-only proteins Puma and Bim are rate-limiting for γ-radiation− and
glucocorticoid-induced apoptosis of lymphoid cells in vivo
Miriam Erlacher, Ewa M. Michalak, Priscilla N. Kelly, Verena Labi, Harald Niederegger, Leigh
Coultas, Jerry M. Adams, Andreas Strasser and Andreas Villunger
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