|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2272-2282
By
From the Department of Pathology and Developmental Biology, Stanford
University School of Medicine, Stanford, CA.
A new transgenic mouse has been generated in which the
proto-oncogene BCL-2 is ubiquitously overexpressed. H2K-BCL-2
transgenic mice overexpress BCL-2 in all cells of the hematolymphoid
system and have been used to assess the role of BCL-2 in protecting
cells of the hematolymphoid system from the consequences of ionizing radiation. We have expanded on previous studies that have demonstrated protection for specific (lymphoid) cell populations and show that systemic overexpression of BCL-2 can protect the hematopoietic system
as a whole, including hematopoietic stem cells (HSC), thus increasing
the radioresistance of the animal. The increase in radioresistance in
H2K-BCL-2 transgenic mice has two components: an increase in
the radioresistance of individual cells and, to a lesser extent, an
increase in the size of certain critically important cell populations,
such as HSC. Bone marrow transplantation experiments show that the
increased radioresistance of the transgenic animals is provided by
cells of the hematopoietic system. Protection against the consequences
of irradiation is not limited to the increased expression levels of
BCL-2 in transgenic mice; levels of endogenous BCL-2 are higher in
lymphocyte populations that survive irradiation in wild-type mice. We
show that ubiquitous overexpression of BCL-2 in the hematopoietic
system can be used to increase the resistance of animals to lethal
challenges such as irradiation.
Bcl-2 IS THE FIRST
characterized member of a family of genes that play a role in the
regulation of apoptosis. Genes from this family fall into two distinct
groups; they prevent or induce apoptosis. The biochemical basis for
their actions remains unclear, despite recent advances in understanding
the way in which the proteins involved in apoptosis act1,2
and interact.3,4 The various members of the Bcl-2
family can form (hetero)dimers and thus can neutralize each others
actions.5 Overexpression of either an apoptosis-preventing
or apoptosis-promoting family member will increase or decrease the
resistance to apoptosis by binding to endogenously expressed
Bcl-2 family members. More recently, it has become clear that
these proteins are also subjected to regulation by posttranslational
modifications, most notably serine phosphorylation.6,7
Overexpression of Bcl-2 in transgenic mice has been studied
extensively in the lymphoid system,8-10 and to a lesser
extent in the myeloid system.11 In addition, Bcl-2
has been overexpressed in several nonhematopoietic tissues in
transgenic mice, including liver,12 neuronal
tissue,13 the olfactory system,14 and the
gonads.15 Although it has been overexpressed in early
hematopoietic progenitor cells in vitro,16 Bcl-2
overexpression has not been targeted to HSCs in vivo. To achieve this,
we used the well-characterized H-2Kb
promoter,17,18 expressed at high levels on
HSCs.19,20 H2K-BCL-2 transgenic mice overexpress
BCL-2 in HSCs, but also in all other hematolymphoid cells, and
presumably in all tissues expressing MHC class I.
Overexpression of Bcl-2 family members can block many
forms of apoptosis, both in transfected cell lines and in transgenic mice. Irradiation is one of the apoptosis-inducing stimuli that can be
blocked by Bcl-2 or Bcl-XL overexpression.
This has been shown in lymphoid-targeted overexpression in transgenic
mice, both in vitro and in vivo.9,21-23 Bcl-2 null
mutant mice show an increased susceptibility to the effects of
irradiation on lymphoid populations in vitro.24,25
Hematopoietic recovery after radiation requires proliferation and
differentiation of HSC into the various depleted compartments. Even
though HSC seem to be less sensitive to the effects of ionizing
irradiation than more mature progenitors26 and lymphoid
cells,27 it is the sensitivity of HSC that limits the
long-term survival, thereby limiting the application of radiation as a
therapeutic tool.28
We have used the ubiquitous expression pattern of the H2K-BCL-2
transgene to address the role of BCL-2 systematically in the response
to ionizing irradiation of hematopoietic cells in vivo. We have found
that not only transgenic lymphoid cells, but also erythroid and myeloid
cells, can tolerate increased levels of irradiation before undergoing
apoptosis, and that this is true both for mature cells and for
progenitor and stem cells. This leads to a dramatically increased
tolerance to radiation for the organism as a whole. In addition we
examined the role of endogenous BCL-2 in radioresistance by comparing
expression levels of lymphoid populations before and after irradiation.
Wild-type (WT) cells that survive irradiation expressed significantly
higher levels of BCL-2 than the pre-irradiated populations.
Molecular biology.
The H2K-i-LTR cassette vector expresses cDNAs under control of the
H2K-promoter/enhancer and Moloney MuLV enhancer/poly(A) site and
consists of the HindIII-Nru I H2Kb
promoter fragment. The Nru I site was converted with a linker into a Not I site, and the fragment was attached to the
Not I-Kpn I fragment of the H2K gene. The
Not I-Kpn I fragment contains intron and exon
sequences (the sites are in exons 1 and 3, respectively). This deletes
the Nru I-Not I fragment of H2K (most of exon 1, including the ATG). The HindIII-Kpn I H2K sequence was
linked to the Eag I-Sal I-fragment from plasmid
pTDK90,29 which contains the Moloney LTR. The human
BCL-2 cDNA-fragment11 was excised from its vector
by digesting with Kpn I and Cla I. The resulting fragment was blunted, Not I linkers were ligated onto it and it was introduced as an Not I fragment into the H2K-i-LTR cassette resulting in the pH2K-BCL-2. Plasmids were constructed and
tested according to established procedures.30 RNA was
isolated and analyzed as described.31 The probe used was
the BCL-2 fragment contained in the transgene.
Mice.
The HindIII fragment of pH2K-BCL-2 that was used for
microinjection was isolated by agarose gel electrophoresis,
electroeluted, and treated with Geneclean (BIO101, Vista, CA) according
to the manufacturer's instructions. The DNA was injected into zygotes from crosses between F1(C57BL/6 × C3H) mice. Mice that were
positive by Southern blot analysis were back-crossed with C57BlKa,
Thy-1.1, Ly-5.1 (H-2b). The transgene was followed in later
generations by flow cytometric screening for expression of human BCL-2
protein in peripheral blood cells.
Irradiation.
Mice, and purified HSCs, were irradiated with a 200-kV x-ray machine.
The dose rate is 63 cGy/min. Fractionated irradiation was done at
3-hour intervals. Cells were kept on ice during irradiation. Mice were
given acid water before irradiation and antibiotic water (1.1 g/L
neomycin sulfate and 106 U/L polymyxin B sulfate) for at
least 8 weeks after irradiation to reduce the chance of infection from
opportunistic pathogens. Mice used for irradiation were 8 to 12 weeks
old.
Tissue culture.
Bone marrow-derived mast cells (BMMC) were cultured as
described.32 Briefly, bone marrow cells were plated in RPMI
1640 with 15% fetal calf serum (FCS), 5 × 10 Flow cytometry.
Single-cell suspensions were obtained in staining medium consisting of
phosphate-buffered saline (PBS) plus 3% FCS and 10 mmol/L Hepes, pH
7.0, after ammonium chloride lysis of the red blood
cells,34 if necessary, and straining through a nylon mesh. Cells were preincubated with mouse-IgG (Sigma, St Louis, MO) and stained for 20 minutes with the various antibodies at the appropriate dilutions and, if necessary, incubated with secondary antibodies. BCL-2
stainings were modified from Veis et al.35 Cells were stained with antibodies against surface markers, followed, when staining for the human BCL-2 protein, by fixation (5 minutes 0.8% formaldehyde in PBS) and permeabilization (5 minutes 0.3% saponin in
staining medium). Cells were then incubated for 3 to 16 hours with
anti-BCL-2 antibody in 0.3% saponin in staining medium, followed, if
necessary, by incubation with the appropriate secondary antibody for 20 minutes. For staining of the mouse BCL-2 protein, the fixation step was
omitted, and cells were incubated with the BCL-2 antibody for 2 hours
in 0.03% saponin in staining medium. Sorting of HSC was done as
described by Morrison and Weissman.34 Briefly, bone marrow
cells are enriched for Sca-1+ cells, using
MACS-columns (Milteny Biotech, Auburn, CA). The stained
(Thy1.1-FITC, Lineage-cocktail, Sca-1-biotin and c-KIT-APC, followed by
the secondary antibodies anti-rat-PE and streptavidin-texas red) and
enriched cells are sorted as described. The lineage cocktail consists
of antibodies against CD3, CD4, CD5, CD8, B220, TER119, Mac-1, and
Gr-1. Labeled cells were analyzed and sorted with a dual laser
fluorescence-activated cell sorter (FACS) (Becton Dickinson Immunocytometry Systems, San Jose, CA), modified as described by Parks
et al,36 and made available through the FACS shared user
group at Stanford University. Dead cells were excluded from analysis by
their propidium iodide staining characteristics. Two parameter data are
presented as 5% probability plots.
Western blot analysis.
Organs were lysed in KLB-buffer (1% Triton X100, 0.05% sodium dodecyl
sulfate [SDS] 150 mmol/L NaCl, 5 mmol/L EDTA, and 10 mmol/L
Na-phosphate, pH 7.2). The lysates were cleared by centrifugation (10 minutes, microfuge) and the protein content was determined using the
Bio-Rad protein assay, according to the manufacturer's instructions
(Bio-Rad Laboratories, Richmond, CA). The proteins were separated by
SDS-polyacrylamide gel electrophoresis (PAGE) and blotted onto
nitrocellulose as described.37 The dried blots were
prehybridized with 5% milk powder in PBS, followed by incubation with
a mouse-anti-human BCL-2 antibody (Dako). Staining was visualized using an ECL-kit (Amersham, Arlington Heights, IL) according to the
manufacturer's instructions.
H2K-BCL-2 transgenic mice express functional protein.
After introduction of the H2K-BCL-2 construct (Fig
1A) into zygotes, 64 mice were obtained, 10 of which carried the transgene. Analysis has concentrated on
founderlines 1038, 1043, and 1053. Southern blot analysis showed low to
intermediate copy numbers for these founderlines. The percentage of
positive founder animals with high expression levels (see below)
indicates that there is no selection against ubiquitous overexpression
of this transgene during development.
Radioresistance of H2K-BCL-2 transgenic mice.
Overexpression of Bcl-2 protects cells from death induced by
irradiation. In view of the ubiquitous expression of this transgene, which includes expression in progenitor and stem cells, we tested whether H2K-BCL-2 transgenic mice show an increased resistance to the lethal effect of irradiation. As shown in Fig
3A, H2K-BCL-2 transgenic mice had
an increased resistance to total body irradiation (TBI); the
LD50/30 (dose at which 50% of the animals survive for at
least 30 days) for single-dose irradiation has increased from approximately 6.5 Gy (WT) to 8.5 Gy (H2K-BCL-2). Whereas all
animals, transgenic and WT, die after receiving doses higher than 9.5 Gy, death is delayed in transgenic animals. WT animals die at day 9 or
10 after receiving 12 Gy TBI; H2K-BCL-2 mice die 3 to 4 days later. After receiving 15 Gy total body irradiation (TBI), WT animals
die at days 5 to 6, and transgenic mice 1 to 3 days later. More than
80% of the transgenic mice survive long term (more than 3 months) when
subjected to a routine lethal preconditioning regimen for bone marrow
or stem cell transplantation, fractionated irradiation of 9.5 Gy (two
doses of 4.75 Gy, 3 hours apart; Fig 3B). Radiation-induced death at
this dose is caused by failure of the hematopoietic system. This is
confirmed by bone marrow transfer experiments: bone marrow cells
(5 × 105) from either H2K-BCL-2 transgenic mice
(H-2Kb) or WT littermates were transferred into lethally
irradiated (split-dose, 8 Gy) allogeneic host mice (Balb/c,
H-2Dd). Multilineage, long-term reconstitution was
confirmed by analysis of circulating white blood cells, using MHC class
I to distinguish host and donor-derived cells. Reconstituted mice were
reirradiated (lethal dose, 9.5 Gy, split-dose) 3 to 4 months after
reconstitution. As expected, none of the nonreconstituted mice, or mice
that were reconstituted with WT bone marrow, survived (Fig 3C).
However, four of five mice that were repopulated with H2K-BCL-2
bone marrow from founderlines 1038 or 1039 survived this second TBI.
This is significantly different (p = .0238, Fisher's
exact test) from the expected outcome of lethal irradiation
Effects of irradiation on the hematopoietic system.
Lethal irradiation has profound effects on the hematopoietic system. In
less than a day the number of cells, and composition of the
hematopoietic organs, changes dramatically. Total cellularity of the
hematopoietic organs in WT mice is reduced by two to three orders of
magnitude in a few days (Fig
4A).
Cellularity continues to decrease over time until the animal dies,
usually 12 to 14 days after irradiation. Severe infections, or
irradiation doses of 15 Gy or higher that lead to extensive damage to
the gastrointestinal tract, can lead to earlier deaths (5 to 8 days
after irradiation). The relative reduction in cellularity after
irradiation is far less severe in H2K-BCL-2 transgenic mice,
one to two orders of magnitude less (Fig 4A). Cellularity is shown as a
percentage of the unirradiated organ, to illustrate the difference in
radiosensitivity of the cells. It does not take into account the
increased size of spleen and thymus in transgenic animals (Table 1).
The absolute difference in size of splenic and thymic cell populations
in transgenic mice thus is two to three times larger than indicated in
(Fig 4A and C). Such a correction is not necessary for bone marrow, for
which the cell counts do not differ between transgenic and WT animals.
Protection against radiation-induced death is extended to all
hematolymphoid cell types (Fig 4B and C). Most apparent is the effect
on B and pre-B lymphocytes, which, in WT mice, virtually disappear from
bone marrow within a day. In H2K-BCL-2 transgenic mice large
numbers of B220+ cells were retained in both bone marrow
and spleen, and their numbers begin to increase again after 9 days.
Nucleated erythroid precursors (TER119+) and myeloid cells
(Mac-1+) also remain in much larger numbers (Fig 4C). The
composition of WT bone marrow changes drastically after irradiation,
the myeloid cells (Gr-1 and Mac-1+ cells40)
(Fig 4B), which are more radioresistant, initially make up the vast
majority of cells. At later points, a large percentage of NK-1.1 cells
can be found (see below). By contrast, in H2K-BCL-2 transgenic
bone marrow, all major populations
Quantitative effects of irradiation on hematopoietic progenitors and
stem cells.
Clonogenic hematopoietic progenitors are sensitive to the effects of
irradiation, albeit to different degrees for different cells.26 We have tested the effects of irradiation on
progenitors from H2K-BCL-2 transgenic and WT littermate mice by
evaluating two different populations: (1) clonogenic precursors,
defined by colony formation on AC-6 or AC-11 stroma
cells33; and (2) sorted HSC, using colony-forming
unit-spleen (CFU-S) day 12 as an assay. Results obtained
from plating on AC6-2.1 and AC11 cells did not differ significantly and
have been combined. In this assay, comparable to various colony-forming
cell (CFC) assays in semisolid media, approximately 1% of
nonirradiated bone marrow gave rise to colonies of B-lymphoid,
erythroid, and myeloid cells, confirmed by flow cytometry (data not
shown). In this experiment, whole bone marrow was plated, isolated
either from nonirradiated mice or from mice that had been irradiated
immediately before plating. The plating efficiency of nonirradiated
bone marrow on AC stroma cells was similar in WT and transgenic
cultures (Fig 5A). At low irradiation
doses, cells from both WT and transgenic mice were able to repair
damage, as indicated by the shoulders in the curves. At higher doses,
an exponential killing curve is seen. However, curve-fitting shows that
transgenic bone marrow is less susceptible to radiation induced death;
the slopes of these curves differ significantly, P = .0026. The
D0 value (dose that kills 1 log e, or 63%, of cells at the
exponential phase) increases from .55 Gy (WT) to 1.20 Gy
(H2K-BCL-2), the extrapolation number n decreases from 100 to
3.3. The corresponding D10 values (dose that kills 1 log10,
or 90%, of cells) were 0.92 Gy for WT and 1.77 Gy for
H2K-BCL-2. Qualitative shifts occur after irradiation. At a
4-Gy dose, the percentage of B lymphocytes after 8 days on AC6 is 5.7 times higher (41.8% v 7.3%) in H2K-BCL-2 transgenic than in WT cultures. WT cultures contain relatively more myeloid cells,
89.1% versus 44.9% in transgenic cultures (average of two experiments). We also analyzed the radiation sensitivity of purified populations of HSC (Sca-1hi c-KIThi
Thy-1.1lo Linneg/lo), isolated by
FACS. Day 12 CFU-S was used as an assay for sorted HSC after
irradiation, because this is a quantitative assay for an activity
present in purified populations of HSC. We found that these cells did
not have a discernible repair phase (Fig 5B). Purified
H2K-BCL-2 transgenic HSCs have an increased resistance to the
effects of irradiation, and the slopes of the response curves obtained
differ significantly, P = .0018. D0 values were 0.70 Gy for the WT HSCs and 1.18 Gy for H2K-BCL-2 HSCs. The
corresponding D10 values are 1.63 Gy for WT and 2.74 Gy for
H2K-BCL-2 HSCs. The inherent difference in radiosensitivity
between WT and H2K-BCL-2 HSC was confirmed by a mixing
experiment. A mixture of HSC (Sca-1hi c-KIThi
Thy-1.1lo Linneg/lo) was isolated from WT
(66%) and H2K-BCL-2/1038 (33%) bone marrow, irradiated at 0, 1.3, or 5.1 Gy, and injected into lethally irradiated hosts, 100 or 200 cells per animal (0 Gy), 500 cells per animal (1.3 Gy) or 2,000 cells
per animal (5.1 Gy). Host and donor cells can be distinguished by the
congenic Ly5.1/Ly5.2 marker, WT, and H2K-BCL-2 cells by
expression of human BCL-2 protein. Reconstituted animals were killed at
day 12, and the spleens were pooled and analyzed by flow cytometry. As
expected, the spleens contained myeloid and erythroid (Gr-1, Mac-1,
TER119), but no lymphoid (B220, CD3, NK-1.1) donor-derived cells
(results not shown). However, the percentage of donor-derived myeloid
cells derived from transgenic HSC increased from 32% (nonirradiated
HSC, 7 animals) to 58% (1.3 Gy irradiation, 5 animals) to 96% (4 Gy
irradiation, two animals).
Endogenous Bcl-2 is expressed at higher levels after
irradiation.
Because overexpression of a BCL-2 transgene confers an advantage to
cells that have been irradiated, we investigated expression of
endogenous BCL-2 in cell populations before and after irradiation in WT
mice. The radioresistant lymphocyte populations that survive irradiation express higher levels of the endogenous BCL-2 protein than
phenotypically similar unirradiated populations (Fig
6). These higher levels of the endogenous
protein after irradiation are not apparent in H2K-BCL-2
transgenic mice. However, in transgenic mice, expression of the
transgene is higher in cells surviving irradiation. Higher levels of
endogenous BCL-2 after irradiation are not seen in all types of
hematopoietic cells. Some myeloid cells (eg, Mac-1-positive cells in
bone marrow) do not show altered expression levels in cells that
survive irradiation.
We have a created a transgenic mouse model in which the human
BCL-2 gene is overexpressed in cells from all hematopoietic lineages, including progenitor and stem cells. Surprisingly, transgenic animals develop normally, except for an expanded lymphoid compartment. Interestingly, the expanded lymphoid compartment in these transgenics includes the thymus, something that has not been observed in other transgenic mice that overexpress BCL-2 in T
cells.9,10,38 Reasons for this difference could be that
this transgene is expressed earlier during thymic T-cell
differentiation, or that overexpression in H2K-BCL-2 transgenic
mice extends to the non-T-cell compartments in the thymus, allowing
them to expand and support increased T-cell development. In agreement
with high expression of functional BCL-2 in T cells is the fact that
the H2K-BCL-2 transgene is able to rescue T-cell development in
IL-2R Submitted August 21, 1997;
accepted November 13, 1997.
We thank Libuse Jerabek for laboratory management, Julie Christensen
and Andreea Nicoleau for the generation of transgenic mice, Veronica
Braunstein for antibody preparation, Lucino Hidalgo for animal care,
Tim Knaak for assistance in fluorescence-activated cell sorting, Eric
Lagasse for performing the apoptosis assays on monocytes and
macrophages, Annette Schlageter for critically reading the manuscript,
and Mark Alkema for providing the pTDK cassette vector.
1.
Yang J,
Liu X,
Bhalla K,
Kim CN,
Ibrado AM,
Cai J,
Peng T-I,
Jones DP,
Wang X:
Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked.
Science
275:1129,
1997
2.
Kluck RM,
Bossy-Wetzel E,
Green DR,
Newmeyer DD:
The release of cytochrome from mitochondria: A primary site for Bcl-2 regulation of apoptosis.
Science
275:1132,
1997
3.
Chinnaiyan AM,
O'Rourke K,
Lane BR,
Dixit VM:
Interaction of CED-4 with CED-3 and CED-9: A molecular framework for cell death.
Science
275:1122,
1997
4.
Wu D,
Wallen HD,
Nunez G:
Interaction and regulation of subcellular localization of CED-4 by CED-9.
Science
275:1126,
1997
5.
Oltvai ZN,
Korsmeyer SJ:
Checkpoints of dueling dimers foil death wishes.
Cell
79:189,
1994[Medline]
[Order article via Infotrieve]
6.
Zha J,
Harada H,
Yang E,
Jockel J,
Korsmeyer SJ:
Serine phosphorylation of death agonist BAD in response to survival factors results in binding to 14-3-3 not BCL-XL.
Cell
87:619,
1996[Medline]
[Order article via Infotrieve]
7.
Wang H-G,
Rapp UR,
Reed JC:
Bcl-2 targets the protein kinase Raf-1 to mitochondria.
Cell
87:629,
1996[Medline]
[Order article via Infotrieve]
8.
McDonnell TJ,
Deane N,
Platt FM,
Nunez G,
Jaeger U,
McKearn JP,
Korsmeyer SJ:
bcl-2-Immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation.
Cell
57:79,
1989[Medline]
[Order article via Infotrieve]
9.
Strasser A,
Harris AW,
Cory S:
bcl-2 Transgene inhibits T cell death and perturbs thymic self-censorship.
Cell
67:889,
1991[Medline]
[Order article via Infotrieve]
10.
Sentman CL,
Shutter JR,
Hockenberry D,
Kanagawa O,
Korsmeyer SJ:
bcl-2 Inhibits multiple forms of apoptosis but not negative selection in thymocytes.
Cell
67:879,
1991[Medline]
[Order article via Infotrieve]
11.
Lagasse E,
Weissman I:
bcl-2 Inhibits apoptosis of neutrophils but not their engulfment by macrophages.
J Exp Med
179:1047,
1994
12.
Lacronique V,
Mignon A,
Fabre M,
Viollet B,
Rouquet N,
Molina T,
Porteu A,
Henrion A,
Bouscary D,
Varlet P,
Joulin V,
Kahn A:
Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice.
Nature Med
2:80,
1996[Medline]
[Order article via Infotrieve]
13.
Farlie PG,
Dringen R,
Rees SM,
Kannourakis G,
Bernard O:
bcl-2 Transgene expression can protect neurons against developmental and induced cell death.
Proc Natl Acad Sci USA
92:4397,
1995 |
Abstract
Introduction
Abstract
5 mol/L
-mercaptoethanol, penicillin, streptomycin, and 20% WEHI-3b conditioned medium as a source of IL-3. Nonadherent cells were subcultured weekly. The histology of the mast cells in culture after 4 weeks was checked on May-Grünwald/Giemsa-stained cytospins. For
interleukin-3 (IL-3) deprivation experiments, cells were washed several
times in medium without IL-3 and then incubated in medium without IL-3.
Cells were counted using an improved Neubauer hemocytometer, viable
cells are defined as trypan-blue excluding cells. Splenocytes (1 to
2 × 107) were plated in 10 mL RPMI 1640 with 5% FCS, 5 × 10
untranslated region of BCL-2. Figure 
no 30-day
survivors.
-chain null mutant mice.