|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2272-2282
Systemic Overexpression of BCL-2 in the Hematopoietic System Protects
Transgenic Mice From the Consequences of Lethal Irradiation
By
Jos Domen,
Kimberly L. Gandy, and
Irving L. Weissman
From the Department of Pathology and Developmental Biology, Stanford
University School of Medicine, Stanford, CA.
 |
ABSTRACT |
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.
| |
INTRODUCTION |
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.
| |
MATERIALS AND METHODS |
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 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 × 105 mol/L -mercaptoethanol, penicillin,
streptomycin in T25 flasks. At regular intervals, samples were taken
and the viability of the cells determined, or cells were analyzed by
flow cytometry as described below. AC6-2.1 murine bone marrow stroma
cells33 were maintained in the same medium as splenocytes.
Cells were subcultured using collagenase/dispase (Boehringer, Mannheim,
Germany). Cells were irradiated before plating of bone marrow cells
(3,000 rads, Cs source). Bone marrow from irradiated mice was plated (102 to 107 cells/well on 12-well dishes,
depending on irradiation dose). Medium was changed weekly (biweekly
initially for cells plated at high densities).
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.
The rat antibodies 53-7.3 (anti-CD5), 53-6.7 (anti-CD8), TER-119
(anti-erythro), GK 1.5 (anti-CD4), KT 31.1 (anti-CD3), 6B2 (anti-B220),
M1/70 (anti-Mac-1), 8C5 (anti-GR-1), and E13-161 (anti-Sca-1) were
prepared from the respective hybridoma clones as were the conjugates KT
31.1-PE, 6B2-FITC, M1/70-FITC, 8C5-PE, 19XES-FITC (anti-Thy-1.1),
53-2.1-FITC (anti-Thy-1.2), A201.7-APC (anti-Ly5.1) and 2B8-APC
(anti-c-kit). Secondary antibodies were obtained
from Caltag (Burlingame, CA). Avidin Texas red was
obtained from Cappel, anti-human BCL-2 (clone 124) from Dako (Glostrup, Denmark). Anti-mouse BCL-2 (clone 3F11), anti H-2Kb-PE
(clone AF6-88.5), anti H-2Dd-biotin (clone 34-5-8S) and
anti-NK-1.1-PE (clone PK136) were purchased from
Pharmingen (San Diego, CA).
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.
| |
RESULTS |
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.
View larger version (57K):
[in this window]
[in a new window]
| Fig 1.
Expression of the H2K-BCL-2 transgene. (A) The
H2K-BCL-2 transgenic construct (B) Northern blot analysis of
organs from an H2K-BCL-2/1043 transgenic mouse and a wild-type
littermate. The blot is probed with the human BCL-2 cDNA
insert. Kid, kidney; Liv, liver; Spl, spleen; Thy, thymus; Lun, lung;
LNP, peripheral lymph nodes; Hea, heart. The position of the ribosomal
bands is indicated on the right. (C) Western blot analysis of organs
from an H2K-BCL-2 transgenic mouse. The blot is probed with a
human BCL-2-specific antibody. All lanes contain the same amount of protein except lymph nodes, which contains one half the amount of the
other lanes. (D) Analysis of BCL-2 expression in hematopoietic stem
cells. Bone marrow was stained with antibodies against Thy1.1, Sca-1,
Lin, and BCL-2. Data collected from 200,000 bone marrow cells was used
to show the BCL-2 expression in Linneg/lo and
Linneg hematopoietic stem cell populations. Gray histograms
show staining for the human BCL-2 protein in wild-type cells, the bold
lines depict staining in H2K-BCL-2 transgenic cells. A
representative experiment out of three is shown.
|
|
RNA analysis of founderline 1043 (Fig 1B) shows abundant RNA expression
in spleen thymus, lung and kidneys; the lowest expression levels are in
liver. The lower band in Fig 1B most likely represents a shortened RNA
transcript attributable to cryptic polyadenylation sites in the 3
untranslated region of BCL-2. Figure 1C shows BCL-2 protein
levels in different organs. These results are consistent with the RNA
data; BCL-2 is weakly expressed in the liver, whereas it is highly
expressed in hematopoietic organs such as thymus and spleen. The
highest expression level is in the lymph nodes. There are no major
differences in the expression pattern of the BCL-2 protein in the four
lines tested by Western blot analysis: 1038, 1039, 1043, and 1053. Minor differences in expression levels were found in thymus (highest
level detected in 1043) and heart (highest level detected in 1053).
BCL-2, as detected by flow cytometry, was expressed at similar levels
in all nucleated blood cells in the founderlines used for analysis.
As expected, all nucleated bone marrow cells stain positive for the
transgene-derived human BCL-2 protein. Expression in HSCs was
determined in two ways. Flow cytometric analysis of bone marrow stained
for Sca-1, Lin, Thy-1.1, and hBCL-2 (Fig 1D) shows that the
Thy-1.1lo Sca-1hi Linneg/lo HSC
populations express the transgene at high levels, similar to
unfractionated bone marrow. This is true for both the population that
contains many short-term multilineage-reconstituting cells (Linneg/lo, .05% of bone marrow) and the population that
is highly enriched for long-term multilineage reconstituting potential
(Linneg, .01% of bone marrow).34 In addition,
sorted WT and transgenic HSCs characterized by the staining profile
that we routinely employ for HSC isolation (Sca-1hi
c-KIThi Linneg/lo
Thy1.1lo),34 were restained with a human BCL-2
specific antibody and analyzed by flow cytometry and
immunohistochemistry. We found again that all transgenic HSCs express
high levels of human BCL-2 (not shown).
Figure 2A shows the dramatically increased
survival in cultures without added growth factors of splenocytes from
three different founderlines, compared with their WT littermates. Both
B and T lymphocytes are protected. T cells, however, are protected to a
higher degree, as illustrated by the reversed ratio of T versus B cells
after 2 months in culture (FACS plots in Fig 2A). Figure 2B illustrates
survival of bone marrow-derived mast cells after IL-3 withdrawal.
Protection against apoptosis induced by growth factor deprivation has
also been shown in NK-1.1+ splenocytes, lipopolysaccharide
(LPS)-stimulated splenocytes, thymocytes, monocytes, and macrophages
(data not shown).
View larger version (37K):
[in this window]
[in a new window]
| Fig 2.
The H2K-BCL-2 transgene protects cells from
apoptosis. (A) Survival of unstimulated splenocytes from
H2K-BCL-2 transgenic mice (thick lines) and wild-type
littermates (thin lines) in vitro. The transgenic mice were from
founderlines 1038, 1039, and 1053. Data from five animals per group,
cultured in four separate experiments. The FACS plots show the B
(B220+) and T (CD3+) cell populations in
transgenic spleen at day 0, 20, and 62 of culture and in the wild-type
spleen at day 9. (B) Survival of bone marrow-derived mast cells in
vitro from H2K-BCL-2/1043 transgenic mice (thick lines) and
wild-type littermates (thin lines) after IL-3 withdrawal. The cell
culture results from two animals per group are depicted.
|
|
H2K-BCL-2 transgenic mice develop normally, except for a
twofold to fivefold increase in size of the lymphoid organs (thymus, spleen, lymph nodes), in the number of circulating white blood cells,
and in the number of HSCs. Other organs, and the body weight of the
mice, do not significantly differ from WT littermates. Table
1 shows the combined data for different
founder lines. The increase in thymus size and cellularity seen in
H2K-BCL-2 transgenic mice has not been reported in other
transgenic lines that overexpress BCL-2 in the
thymus.9,10,38 Table 2 shows the sizes of the various major subpopulations in the thymus, as defined
by CD4/CD8 and CD3/c-KIT. Although all have expanded, the largest
relative expansion is seen in the c-KIT+ cells that are
undergoing positive selection.39 The
CD3+c-KIT+ cells, which are making the
transition from double positive to single positive cells,39
have expanded the most, approximately 10-fold, in these transgenic
mice. In addition, there is a relative increase in CD4 and CD8 single
positive cells. Thymus expansion (weight) is more pronounced in
founderline 1043 (235% of WT, n = 4) than in founderline 1038 (142%
of WT, n = 5). In addition to the increase in number of lymphoid
cells, there are more HSCs. The number of cells with the surface
phenotype Sca-1hi c-KIThi Thy1.1lo
Linneg/lo in bone marrow is increased twofold to threefold
in transgenic mice.
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 no 30-day
survivors.
View larger version (18K):
[in this window]
[in a new window]
| Fig 3.
Radioprotective effect of the H2K-BCL-2
transgene. (A) Survival of H2K-BCL-2 transgenic and wild-type
mice after single dose irradiation at the doses indicated. Graph is
based on survival data from 84 wild-type and 51 transgenic mice, 2 (extreme values) to 17 (mid-range) mice were assayed per irradiation
dose. The 30-day survival of wild-type mice differs significantly from
transgenic mice at 6.5, 7, and 8 Gy (P values [Fisher's exact
test] are .0359, .0002, and .0310, respectively). (B) Long-term
survival of H2K-BCL-2 transgenic mice after lethal total body
irradiation, 9.5 Gy, split dose. The difference in 90-day survival is
highly significant, P < .0001 (Fisher's exact test). (C)
Survival of reconstituted Balb/c mice after split dose 9.5-Gy
irradiation. These mice were reconstituted with bone marrow from
H2K-BCL-2 transgenic mice and wild-type littermates at 3 to 4 months before 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 populationsmyeloid, lymphoid, and
erythroidare retained. The flow cytometry plots shown in Fig 4B
illustrate that both CD4 and CD8 positive T cells from the spleen are
protected by this transgene. The change in their ratio that is observed
in WT mice (CD8+ cells are far more radiosensitive than
CD4+ cells27) is virtually absent in transgenic
mice. Spleen-derived B lymphocytes from H2K-BCL-2 transgenic
mice also show a dramatically increased radioresistance, as do natural
killer (NK) cells (Fig 4C). The decrease in the number of NK cells is
up to 100-fold less in transgenic mice following irradiation (absolute
numbers up to 300-fold higher). In thymus the most dramatic effect is seen with the most radiosensitive population,
CD4+CD8+ double-positive cells.27
These cells show a 1,000-fold higher survival in transgenic mice
compared with WT mice (Fig 4A and B). Cellularity increases transiently
9 days after irradiation in WT thymus (observed in thymi from 4 of 4 WT
mice, in two experiments). This very rapid increase consists of cells
that are double positive for both CD4 and CD8, presumably derived from
surviving thymic progenitor cells
(CD483 or
CD4lo83).41 It
does not herald stable repopulation, as thymic cellularity collapses
again during the time the mice succumb to the effects of irradiation
(12 to 14 days after irradiation). This induction of
CD4+CD8+ cells after irradiation is reminiscent
of what has been reported in RAG-deficient mice.42
View larger version (64K):
[in this window]
[in a new window]
View larger version (29K):
[in this window]
[in a new window]
| Fig 4.
Radioprotective effect of H2K-BCL-2 for
hematopoietic populations following irradiation. (A) Cellularity in
bone marrow, spleen, and thymus of H2K-BCL-2 transgenic mice
and wild-type littermates following split-dose, 9.5-Gy total
body irradiation. Bold lines, filled symbols depict
H2K-BCL-2, thin lines; open symbols wild-type littermates.
Circles show data from founderline 1043, rectangles from founderline
1053. Each datapoint represents one to three mice, the results from two
separate experiments (circles and rectangles) are shown. (B) Flow
cytometric analysis of bone marrow, thymus and spleen of
H2K-BCL-2/1043 transgenic mice and wild-type littermates before
(day 0) and after split-dose, 9.5-Gy total body irradiation. (Top left)
B cells in bone marrow 16 hours after irradiation. This illustrates the
extreme radiosensitivity of wild-type B cells. (Top right) Myeloid
cells at the same timepoint. These relatively radioresistant cells
accumulate in wild-type mice. (Bottom left) CD4 and CD8 staining in
spleen 6 and 9 days after irradiation, illustrating the relative
radiosensitivity of CD8 cells in wild-type mice. (Bottom right) CD4 and
CD8 populations in thymus 6 and 9 days after irradiation.
(C) Fate of various hematopoietic populations after
irradiation. Symbols as in (A). The top two plots show erythroid cells
in bone marrow (left) and myeloid cells in spleen (right). The second
row shows NK cells (left) and B cells (right) in spleen. All as
percentage of the populations before irradiation.
|
|
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).
View larger version (14K):
[in this window]
[in a new window]
| Fig 5.
Survival of progenitors of H2K-BCL-2 transgenic
and wild-type mice after irradiation. (A) Plating of bone marrow from
irradiated mice on AC-6 or AC11 stromal cells. Bone marrow was isolated
immediately after irradiation, which was given in a single dose, and
plated on preirradiated stroma cells. Plating efficiency of clonogenic cells was determined at day 8 by counting the colonies. Combined data
from three experiments, each datapoint represents one irradiated mouse.
(B) Radioprotection of CFU-S day 12. HSCs were sorted from bone marrow
from H2K-BCL-2 transgenic and wild-type mice, aliquoted, exposed to a single dose of irradiation, and injected into lethally irradiated (9.5 Gy, split dose) C57BI/Ka mice, five mice per group. Spleen colonies were counted on day 12. Error bars indicate standard deviations for each group of five spleens. Combined data from three
separate experiments, containing a total of seven datapoints for the
transgenic, and six for the wild-type mice. Some of these datapoints
overlap and cannot be distinguished in the figure.
|
|
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.
View larger version (33K):
[in this window]
[in a new window]
| Fig 6.
BCL-2 expression in radioresistant populations.
H2K-BCL-2/1038 transgenic and wild-type mice (littermates) were
analyzed for expression of endogenous (mBCL-2) and transgenic (hBCL-2)
protein 2 days after lethal irradiation (9.2-Gy split dose). Staining of CD4+ splenocytes is depicted. Filled gray histogram,
staining in untreated animals; thick line, staining after lethal
irradiation. (Top) Background staining of the secondary antibody
(goat-anti-hamster-FITC) used to detect hamster-anti-mouse BCL-2 or
autofluorescence (mouse-anti-human BCL-2 was FITC-conjugated). (Left)
Mouse BCL-2 expression in a wild-type mouse. (Middle) Mouse BCL-2
expression in a H2K-BCL-2 transgenic mouse. (Right) Expression
of the transgene (human BCL-2) in a H2K-BCL-2 transgenic mouse.
One representative experiment (out of four) is shown.
|
|
| |
DISCUSSION |
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  -chain null mutant mice.43 BCL-2 has been shown to
allow lymphoid cells to survive higher levels of irradiation. We have
used the ubiquitous overexpression of BCL-2 to investigate how
this affects the response to irradiation of multiple hematopoietic cell
populations in transgenic mice. We show here that overexpression of
BCL-2 throughout the hematopoietic system protects mice from
radiation-induced hematopoietic failure, and consequent death. The
hematopoietic populations in H2K-BCL-2 transgenic mice that
have an increased resistance to the effects of irradiation include not
only mature B- and T-cell populations, on which attention has focused
before,9,21-23 but also NK, myeloid, erythroid, progenitor,
and stem cells. The increased radioresistance of more mature
populations is probably important for the short-term survival of
irradiated H2K-BCL-2 transgenic mice, whereas the increased
radioresistance of HSC ensures long-term survival. The increase in
radioresistance provided by the H2K-BCL-2 transgene in mice has
several different components. One of these is an increase in size of
some of the critical cell populations, especially lymphocytes and HSCs,
which means that more cells will survive irradiation. In addition, and
more importantly, there is an increased cellular radioresistance, which
enables cells expressing the transgene to survive higher doses of
irradiation than WT cells, without undergoing apoptosis. It is not
possible to assess directly the contribution of both components to the
increase in HSC survival after irradiation, and thus long-term survival
of the mice, because we cannot generate mice with comparable numbers of
transgenic or WT HSC in a controlled fashion, not even after
transplantation. However, a twofold increase in HSC would only increase
the LD50/30 (based on HSC-survival) of WT mice by 0.48 Gy,
much less than the 2 Gy that is actually observed. It would take
approximately an 18-fold increase in HSC to increase the
LD50/30 by 2 Gy [D0·ln(18), or
D10·log(18)]. Therefore, the increased survival of
transgenic animals cannot be explained by the increase in HSC alone but
is mainly dependent on the increase in cellular radioresistance. The
increased radioresistance of transgenic HSC is demonstrated in an
experiment in which a mixture of WT and transgenic HSC was irradiated
at different doses and injected into lethally irradiated mice; relative
engraftment of H2K-BCL-2 transgenic HSC increases dramatically
with increasing levels of irradiation.
The values that we find for D0 and LD50/30 in
WT mice are consistent both with published values44,45 and
with our own data on HSC in mice. A 2-month-old C57BL/Thy1.1 mouse
contains approximately 1 to 2 × 105 long-term and
short-term HSC; injection of 40 of these cells radioprotects
approximately 50% of the injected mice.34,46 At 6.5-Gy
irradiation, the depletion should be
6.5/D10·log10. Since we find a
D10 of 1.63 Gy for WT HSC, a 4 log10 depletion is expected. This should leave 10 to 20 HSC, close to the number needed
for 50% radioprotection (40 cells), taking injection losses into
account. Because of the difference in D10 in
H2K-BCL-2 transgenic mice, a 6.5-Gy irradiation dose would only
lead to a 2.4 log10 depletion, leaving approximately
40-fold more HSC (not taking into account the increased starting number
of HSC). However, at 8.5 Gy, the observed LD50/30 dose for
transgenic mice, only a 3.1 log10 depletion would be
predicted, not enough to explain the hematopoietic failure observed
(death at 10 to 20 days after irradiation). One explanation could be
that the CFU-S12 measured does not reflect in vivo
radioprotection. More likely, and in line with published
observations,26 would be the possibility that survival of
HSCs does not closely follow exponential kinetics at higher irradiation
doses. The large number of cells that would need to be isolated by
sorting prevents a direct testing of survival at these high doses.
Clonogenic precursors that form colonies on AC6 have lower
D0 and D10 values than sorted HSC. This is
seemingly in contrast to published data,26,47 that show
myeloid-CFC to be at least as radioresistant as stem cells. However, AC
stroma reads out not only myeloid CFC, but CFC with erythroid and
B-lymphoid potential as well. These precursors clearly differ in their
radiosensitivity, with B-lymphoid precursors being the most
radiosensitive. As shown in Fig 5A, a 5.2 log10 depletion
(.000006%, instead of 1% of bone marrow-forming colonies) in
precursors is seen in WT bone marrow at the LD50/30 level
of irradiation (6.5 Gy). This number is directly derived from the graph
shown in Fig 5A and does take into account the repair phase observed at
lower irradiation doses. Assuming approximately 3 × 108
bone marrow cells per mouse,38 and 1% of the bone marrow
cells reading out as CFC on AC stroma cells (Fig 5A), this would
indicate a near-total depletion of committed hematopoietic precursor
cells. In H2K-BCL-2 transgenic bone marrow only a 2.9 log10 depletion is seen at the same irradiation level,
which would leave an estimated 4 × 103 committed
progenitor cells, assuming the same number of clonogenic cells per bone
marrow initially.
Increased levels of endogenous and transgenic BCL-2 are seen in
surviving lymphocyte populations after irradiation. Either cells
expressing BCL-2 at high levels are selected following irradiation, or
irradiation upregulates BCL-2 expression. Analysis of the effects of
irradiation in a primitive human hematopoietic cell line does not
support a direct upregulation as a consequence of
irradiation.48 The fact that endogenous BCL-2 levels remain
the same in transgenic cells after irradiation also suggests that BCL-2
expression is not induced by irradiation. Thus, higher levels of
endogenous BCL-2 correlate with increased radioresistance, analogous to
the protection provided by the transgene-derived protein. Different expression levels of anti-apoptotic genes such as Bcl-2 might explain the observed biphasic nature of the radiosensitivity of T-cell
subsets27 and the PHA-induced radioresistance in
radiosensitive thymocyte subsets.49 Another argument for
the involvement of BCL-2 in the protection against radiation-induced
apoptosis comes from the recent observations that the
protein-serine/threonine kinase Raf-1, which is activated by
upstream protein-tyrosine kinases after irradiation,50 can
directly associate with the Bcl-2 protein and functionally
activate it by phosphorylating,7 and thus
inactivating,6 Bad, a negatively acting member of the Bcl-2 family. While the radioprotective effect of
Bcl-2 could reflect only the increased survival time of cells,
allowing normal DNA repair to take place before the cell attempts to
undergo cell division, recent results suggest a more direct
interaction. It has been reported that overexpression of Bcl-2
can prevent downregulation of DNA repair enzymes such as
apurinic/apyrimidinic endonuclease.51 This implies that
cells overexpressing Bcl-2 might not only survive longer, and
thus have more time to repair DNA damage, but might also have an
increased repair capacity. The fact that higher BCL-2 levels after
irradiation are only seen in lymphoid populations, but not in myeloid
cells, indicates that Bcl-2 levels are limiting in the former,
but not the latter. This is in line with the observation that lymphoid,
but not myeloid cells in Bcl-2 null mutant mice are
hypersensitive to apoptotic stimuli.24,25 For cell
populations in which higher Bcl-2 levels are not seen in cells
that have survived irradiation, other Bcl-2 family members,
such as Bcl-XL or A1, may be critical.
We have shown that ubiquitous overexpression of BCL-2 in
H2K-BCL-2 transgenic mice increased their resistance to
radiation, by protecting the hematopoietic system as a whole.
Surprisingly, this overexpression did not disrupt normal developmental
processes in which apoptosis is thought to play a critical role. The
reason for this is currently under investigation as is the effect of BCL-2 overexpression on HSCs in vivo and in vitro.
Overexpression of an anti-apoptotic protein has obvious advantages for
transformed cells. Irradiation, and most chemotherapeutic agents,
function through induction of apoptosis.52 The results
presented here show that overexpression of Bcl-2 should
increase the resistance to therapeutic interventions such as
irradiation, not only for tumor, but also for a wide range of bystander
cells. It is difficult to assess the contributions of single genes to
the increased resistance of tumor cells to apoptosis induced by
irradiation or chemotherapeutic agents using tumor derived cell lines
that carry many, mostly unknown, mutations. The transgenic mouse model
presented here allows the systematic investigation of the role that
oncogenes such as Bcl-2 can play in overcoming radiation or
chemotherapeutic challenges in both mature and immature hematopoietic
cells of all lineages. Crosses between different transgenic mice will
allow the evaluation of combinations of genes.
The ability of Bcl-2 to block apoptosis induced by a number of
cancer therapeutic agents should lead to a consideration of the
clinical use of Bcl-2 as a gene therapy agent enabling normal hematolymphoid cells to survive ordinarily supralethal therapeutic regimens. The risk/benefit ratio may be unacceptably high, since Bcl-2 is a proto-oncogene, presumably resulting from its
anti-apoptotic functions that allow DNA alterations to accumulate.
However, if means to purge Bcl-2 overexpressing cells
quantitatively in vivo can be achieved, or if its expression can be
regulated (especially downregulated) effectively, the risk/benefit
ratio for several kinds of cancer patients may become acceptable.
| |
FOOTNOTES |
Submitted August 21, 1997;
accepted November 13, 1997.
Supported by grants from the Dutch Cancer Foundation/Koningin
Wilhelmina Fonds (J.D.), American Cancer Society-California Division
(K.L.G.) and SyStemix/Sandoz (I.L.W.).
Address reprint requests to Jos Domen, PhD, Department of Pathology and
Developmental Biology, B263 Beckman Center, Stanford University School
of Medicine, Stanford, CA 94305-5428.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
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.
| |
REFERENCES |
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
4.
Wu D,
Wallen HD,
Nunez G:
Interaction and regulation of subcellular localization of CED-4 by CED-9.
Science
275:1126,
1997[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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/Free Full Text]
14.
Hayward MD,
Morgan JI:
The olfactory system as a model of the contribution of gene expression to programmed cell death.
Chem Senses
20:261,
1995[Abstract/Free Full Text]
15.
Hsu SY,
Lai RJ,
Nanuel D,
Hsueh AJ:
Different 5 flanking regions of the inhibin-alpha gene target transgenes to the gonad and adrenal in an age-dependent manner in transgenic mice.
Endocrinology
136:5577,
1995[Abstract]
16.
Fairbairn LJ,
Cowling GJ,
Reipert BM,
Dexter TM:
Suppression of apoptosis allows differentiation and development of a multipotent hemopoietic cell line in the absence of added growth factors.
Cell
74:823,
1993[Medline]
[Order article via Infotrieve]
17.
Morello D,
Moore G,
Salmon AM,
Yaniv M,
Babinet C:
Studies on the expression of an H-2K/human growth hormone fusion gene in giant transgenic mice.
EMBO J
5:1877,
1986[Medline]
[Order article via Infotrieve]
18.
Breuer M,
Slebos R,
Verbeek S,
van Lohuizen M,
Wientjens E,
Berns A:
Very high frequency of lymphoma induction by a chemical carcinogen in pim-1 transgenic mice.
Nature
340:61,
1989[Medline]
[Order article via Infotrieve]
19.
Bauman JG,
de Vries P,
Pronk B,
Visser JW:
Purification of murine hematopoietic stem cells and committed progenitors by fluorescence activated cell sorting using wheat germ agglutin and monoclonal antibodies.
Acta Histochem
36:241,
1988
20.
Spangrude GJ,
Scollay R:
Differentiation of hematopoietic cells in irradiated mouse thymic lobes.
J Immunol
145:3661,
1990[Abstract]
21.
Griffiths SD,
Goodhead DT,
Marsden SJ,
Wright EG,
Krajewski S,
Reed JC,
Korsmeyer SJ,
Greaves M:
Interleukin 7-dependent lymphocyte precursor cells are ultrasensitive to apoptosis.
J Exp Med
179:1789,
1994[Abstract/Free Full Text]
22.
Grillot DAM,
Merino R,
Nunez G:
Bcl-XI displays restricted distribution during T cell development and inhibits multiple forms of apoptosis but not clonal deletion in transgenic mice.
J Exp Med
182:1973,
1995[Abstract/Free Full Text]
23.
Chao DT,
Linette GP,
Boise LH,
White LS,
Thompson CB,
Korsmeyer SJ:
Bcl-xl and Bcl-2 repress a common pathway of cell death.
J Exp Med
182:821,
1995[Abstract/Free Full Text]
24.
Nakayama K-I,
Nakayama K,
Negishi I,
Kuida K,
Shinkai Y,
Louie MC,
Fields LE,
Lucas PJ,
Stewart V,
Alt FW,
Loh DY:
Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice.
Science
261:1584,
1993[Abstract/Free Full Text]
25.
Veis DJ,
Sorenson CM,
Shutter JR,
Korsmeyer SJ:
Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair.
Cell
75:229,
1993[Medline]
[Order article via Infotrieve]
26.
Down JD,
Boudewijn A,
van Os R,
Thames HD,
Ploemacher RE:
Variations in radiation sensitivity and repair among different hematopoietic stem cell subsets following fractionated irradiation.
Blood
86:122,
1995[Abstract/Free Full Text]
27.
Williams JL,
Patchen ML,
Darden JH,
Jackson WE:
Effects of radiation on survival and recovery of T lymphocyte subsets in C3H/HeN mice.
Exp Hematol
22:510,
1994[Medline]
[Order article via Infotrieve]
28. Shank B: Radiotherapeutic principles of bone marrow
transplantation, in Forman SJ, Blume KG, Thomas ED (eds): Bone Marrow
Transplantation. Boston, MA, Blackwell Scientific, 1994, p 96
29.
Alkema MJ,
van der Lugt NMT,
Bobeldijk RC,
Berns A,
van Lohuizen M:
Transformation of axial skeleton due to overexpression of bmi-1 in transgenic mice.
Nature
374:724,
1995[Medline]
[Order article via Infotrieve]
30. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A
Laboratory Manual, (ed 2). Cold Spring Harbor, NY, Cold Spring Harbor
Laboratory, 1989
31.
Domen J,
von Lindern M,
Hermans A,
Breuer M,
Grosveld G,
Berns A:
Comparison of the mouse and human pim-1 cDNAs: nucleotide sequence and immunological identification of the in vitro synthesized pim-1 protein.
Oncogene Res
1:103,
1987[Medline]
[Order article via Infotrieve]
32.
Domen J,
van der Lugt NMT,
Laird PW,
Saris CJM,
Clarke AR,
Hooper ML,
Berns A:
Impaired IL-3 response in Pim-1 deficient bone marrow derived mast cells.
Blood
82:1445,
1993[Abstract/Free Full Text]
33.
Whitlock CA,
Tidmarsh GF,
Muller-Sieburg C,
Weissman IL:
Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule.
Cell
48:1009,
1987[Medline]
[Order article via Infotrieve]
34.
Morrison SJ,
Weissman II:
The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.
Immunity
1:661,
1994[Medline]
[Order article via Infotrieve]
35.
Veis D,
Sentman CL,
Bach EA,
Korsmeyer SJ:
Expression of the Bcl-2 protein in murine and human thymocytes and in peripheral T cells.
J Immunol
151:2546,
1993[Abstract]
36.
Parks DR,
Hardy RR,
Herzenberg LA:
Three-color immunofluorescence analysis of mouse B-lymphocyte populations.
Cytometry
5:159,
1984[Medline]
[Order article via Infotrieve]
37.
Saris CJ,
Domen J,
Berns A:
The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG.
EMBO J
10:655,
1991[Medline]
[Order article via Infotrieve]
38.
Katsumata M,
Siegel RM,
Louie DC,
Miyashita T,
Tsujimoto Y,
Nowell PC,
Greene MI,
Reed JC:
Differential effects of Bcl-2 on T and B cells in transgenic mice.
Proc Natl Acad Sci USA
89:11376,
1992[Abstract/Free Full Text]
39.
Akashi K,
Weissman IL:
The c-kit-positive maturation pathway in mouse thymic T cell development: Lineages and selection.
Immunity
5:147,
1996[Medline]
[Order article via Infotrieve]
40.
Lagasse E,
Weissman IL:
Flow cytometric identification of murine neutrophils and monocytes.
J Immunol Methods
197:139,
1996[Medline]
[Order article via Infotrieve]
41.
Godfrey DI,
Zlotnik A,
Suda A:
Phenotypic and functional characterization of c-kit expression during intrathymic T cell development.
J Immunol
149:2281,
1992[Abstract]
42.
Guidos CJ,
Williams CJ,
Wu GE,
Paige CJ,
Danska JS:
Development of Cd4+CD8+ thymocytes in RAG-deficient mice through a T cell receptor chain-independent pathway.
J Exp Med
181:1187,
1995[Abstract/Free Full Text]
43.
Kondo M,
Akashi K,
Domen J,
Sugamura K,
Weissman IL:
Bcl-2 rescues T lymphopoiesis, but not B or NK cell development in common chain-deficient mice.
Immunity
7:1,
1997[Medline]
[Order article via Infotrieve]
44. Storer JB: Acute responses to ionizing radiation, in Green EL
(ed): Biology of the Laboratory Mouse (ed 2). New York, NY,
McGraw-Hill, 1966, p 427
45.
McCarthy KF:
Population size and radiosensitivity of murine hematopoietic endogenous long-term repopulating cells.
Blood
89:834,
1997[Abstract/Free Full Text]
46.
Spangrude GJ,
Heimfeld S,
Weissman IL:
Purification and characterization of mouse hematopoietic stem cells.
Science
241:58,
1988[Abstract/Free Full Text]
47.
Meijne EIM,
van der Winden-van Groenewegen RJM,
Ploemacher RE,
Vos O,
David JAG,
Huiskamp R:
The effects of x-irradiation on hematopoietic stem cell compartments in the mouse.
Exp Hematol
19:617,
1991[Medline]
[Order article via Infotrieve]
48.
Clave E,
Carosella ED,
Gluckman E,
Dubray B,
Socie G:
Ionizing radiation effects on the KG1a primitive hematopoietic cell line.
Int J Radiat Oncol Biol Phys
35:709,
1996[Medline]
[Order article via Infotrieve]
49. Strober S, Weissman IL: Immunosuppressive and tolerogenic
effects of whole-body, total lymphoid, and regional irradiation, in
Salaman JR (ed): The Current Status of Modern Therapy, vol 7. Lancaster, UK, MTP, 1981, p 19
50.
Kasid U,
Suy S,
Dent P,
Ray S,
Whiteside TL,
Sturgill TW:
Activation of Raf by ionizing radiation.
Nature
382:813,
1996[Medline]
[Order article via Infotrieve]
51.
Robertson KA,
Hill DP,
Xu Y,
Liu L,
Van Epps S,
Hockenberry DM,
Park JR,
Wilson TM,
Kelley MR:
Downregulation of AP endonuclease (APE) expression is associated with the induction of apoptosis in differentiating myeloid cells.
Cell Growth Diff
8:443,
1997[Abstract]
52.
Hannun YA:
Apoptosis and the dilemma of cancer chemotherapy.
Blood
89:1845,
1997[Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
S. Bandyopadhyay, M. Long, H. Z. Qui, A. T. Hagymasi, A. M. Slaiby, M. A. Mihalyo, H. L. Aguila, R. S. Mittler, A. T. Vella, and A. J. Adler
Self-Antigen Prevents CD8 T Cell Effector Differentiation by CD134 and CD137 Dual Costimulation
J. Immunol.,
December 1, 2008;
181(11):
7728 - 7737.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.P. Chao, J. Seita, and I.L. Weissman
Establishment of a Normal Hematopoietic and Leukemia Stem Cell Hierarchy
Cold Spring Harb Symp Quant Biol,
November 6, 2008;
(2008)
sqb.2008.73.031v1.
[Abstract]
[PDF]
|
|
|
|
|
|
|
P. Cheng, C. A. Corzo, N. Luetteke, B. Yu, S. Nagaraj, M. M. Bui, M. Ortiz, W. Nacken, C. Sorg, T. Vogl, et al.
Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein
J. Exp. Med.,
September 29, 2008;
205(10):
2235 - 2249.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Inoue-Narita, K. Hamada, T. Sasaki, S. Hatakeyama, S. Fujita, K. Kawahara, M. Sasaki, H. Kishimoto, S. Eguchi, I. Kojima, et al.
Pten Deficiency in Melanocytes Results in Resistance to Hair Graying and Susceptibility to Carcinogen-Induced Melanomagenesis
Cancer Res.,
July 15, 2008;
68(14):
5760 - 5768.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Ruau, R. Ensenat-Waser, T. C. Dinger, D. S. Vallabhapurapu, A. Rolletschek, C. Hacker, T. Hieronymus, A. M. Wobus, A. M. Muller, and M. Zenke
Pluripotency Associated Genes Are Reactivated by Chromatin-Modifying Agents in Neurosphere Cells
Stem Cells,
April 1, 2008;
26(4):
920 - 926.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. T. Jensen, C. Boiers, S. Kharazi, A. Lubking, T. Ryden, M. Sigvardsson, E. Sitnicka, and S. E. W. Jacobsen
Permissive roles of hematopoietin and cytokine tyrosine kinase receptors in early T-cell development
Blood,
February 15, 2008;
111(4):
2083 - 2090.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Pilbeam, P. Basse, L. Brossay, N. Vujanovic, R. Gerstein, A. N. Vallejo, and L. Borghesi
The Ontogeny and Fate of NK Cells Marked by Permanent DNA Rearrangements
J. Immunol.,
February 1, 2008;
180(3):
1432 - 1441.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Nemeth, L. Topol, S. M. Anderson, Y. Yang, and D. M. Bodine
Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation
PNAS,
September 25, 2007;
104(39):
15436 - 15441.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Ganapati, H. T. Tan, M. Lynch, M. Dolezal, S. de Vos, and J. C. Gasson
Modeling Notch Signaling in Normal and Neoplastic Hematopoiesis: Global Gene Expression Profiling in Response to Activated Notch Expression
Stem Cells,
August 1, 2007;
25(8):
1872 - 1880.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Ma, M. Shi, J. Li, B. Chen, H. Wang, B. Li, J. Hu, Y. Cao, B. Fang, and R. C. Zhao
Senescence-unrelated impediment of osteogenesis from Flk1+ bone marrow mesenchymal stem cells induced by total body irradiation and its contribution to long-term bone and hematopoietic injury
Haematologica,
July 1, 2007;
92(7):
889 - 896.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. H. Wagner, A. M. Drewry, S. MacMillan, W. M. Dunne, K. C. Chang, I. E. Karl, R. S. Hotchkiss, and J. P. Cobb
Surviving sepsis: bcl-2 overexpression modulates splenocyte transcriptional responses in vivo
Am J Physiol Regulatory Integrative Comp Physiol,
April 1, 2007;
292(4):
R1751 - R1759.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Sugiyama, R. T. Rodriguez, G. W. McLean, and S. K. Kim
Conserved markers of fetal pancreatic epithelium permit prospective isolation of islet progenitor cells by FACS
PNAS,
January 2, 2007;
104(1):
175 - 180.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Woelfel, J. Bixby, M. A. Brehm, and F. K.-M. Chan
Transgenic Expression of the Viral FLIP MC159 Causes lpr/gld-Like Lymphoproliferation and Autoimmunity
J. Immunol.,
September 15, 2006;
177(6):
3814 - 3820.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. P. Bailey, M. Kashyap, L. A. Bouton, P. J. Murray, and J. J. Ryan
Interleukin-10 induces apoptosis in developing mast cells and macrophages
J. Leukoc. Biol.,
September 1, 2006;
80(3):
581 - 589.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. V. Priestley, L. M. Scott, T. Ulyanova, and T. Papayannopoulou
Lack of {alpha}4 integrin expression in stem cells restricts competitive function and self-renewal activity
Blood,
April 1, 2006;
107(7):
2959 - 2967.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Wang, B. A. Schulte, A. C. LaRue, M. Ogawa, and D. Zhou
Total body irradiation selectively induces murine hematopoietic stem cell senescence
Blood,
January 1, 2006;
107(1):
358 - 366.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. L. Kee
Id3 Induces Growth Arrest and Caspase-2-Dependent Apoptosis in B Lymphocyte Progenitors
J. Immunol.,
October 1, 2005;
175(7):
4518 - 4527.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Surdez, B. Kunz, A. J. Wagers, I. L. Weissman, and A. V. Terskikh
Simple and Efficient Isolation of Hematopoietic Stem Cells from H2K-zFP Transgenic Mice
Stem Cells,
October 1, 2005;
23(10):
1617 - 1625.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Yamane, S. J. Dylla, M. Muijtjens, and I. L. Weissman
Enforced Bcl-2 expression overrides serum and feeder cell requirements for mouse embryonic stem cell self-renewal
PNAS,
March 1, 2005;
102(9):
3312 - 3317.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. McNeil Coffield, W. S. Helms, Q. Jiang, and L. Su
G{alpha}13 Mediates a Signal That Is Essential for Proliferation and Survival of Thymocyte Progenitors
J. Exp. Med.,
November 15, 2004;
200(10):
1315 - 1324.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-H. Jang and Y.-J. Surh
Bcl-2 Attenuation of Oxidative Cell Death Is Associated with Up-regulation of {gamma}-Glutamylcysteine Ligase via Constitutive NF-{kappa}B Activation
J. Biol. Chem.,
September 10, 2004;
279(37):
38779 - 38786.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Orelio, K. N. Harvey, C. Miles, R. A. J. Oostendorp, K. van der Horn, and E. Dzierzak
The role of apoptosis in the development of AGM hematopoietic stem cells revealed by Bcl-2 overexpression
Blood,
June 1, 2004;
103(11):
4084 - 4092.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Borghesi, L.-Y. Hsu, J. P. Miller, M. Anderson, L. Herzenberg, L. Herzenberg, M. S. Schlissel, D. Allman, and R. M. Gerstein
B Lineage-specific Regulation of V(D)J Recombinase Activity Is Established in Common Lymphoid Progenitors
J. Exp. Med.,
February 17, 2004;
199(4):
491 - 502.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Karlsson, M. Engstrom, M. Jonsson, P. Karlberg, C. J.H. Pronk, J. Richter, and J.-I. Jonsson
Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptotic Bcl-2 family genes
J. Leukoc. Biol.,
November 1, 2003;
74(5):
923 - 931.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Meng, Y. Wang, G. Van Zant, and D. Zhou
Ionizing Radiation and Busulfan Induce Premature Senescence in Murine Bone Marrow Hematopoietic Cells
Cancer Res.,
September 1, 2003;
63(17):
5414 - 5419.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Ara, M. Itoi, K. Kawabata, T. Egawa, K. Tokoyoda, T. Sugiyama, N. Fujii, T. Amagai, and T. Nagasawa
A Role of CXC Chemokine Ligand 12/Stromal Cell-Derived Factor-1/Pre-B Cell Growth Stimulating Factor and Its Receptor CXCR4 in Fetal and Adult T Cell Development in Vivo
J. Immunol.,
May 1, 2003;
170(9):
4649 - 4655.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S L Preston, M R Alison, S J Forbes, N C Direkze, R Poulsom, and N A Wright
The new stem cell biology: something for everyone
Mol. Pathol.,
April 1, 2003;
56(2):
86 - 96.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Al-Hajj, M. S. Wicha, A. Benito-Hernandez, S. J. Morrison, and M. F. Clarke
From the Cover: Prospective identification of tumorigenic breast cancer cells
PNAS,
April 1, 2003;
100(7):
3983 - 3988.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Herodin, P. Bourin, J.-F. Mayol, J.-J. Lataillade, and M. Drouet
Short-term injection of antiapoptotic cytokine combinations soon after lethal gamma -irradiation promotes survival
Blood,
April 1, 2003;
101(7):
2609 - 2616.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. S. Radomska, D. A. Gonzalez, Y. Okuno, H. Iwasaki, A. Nagy, K. Akashi, D. G. Tenen, and C. S. Huettner
Transgenic targeting with regulatory elements of the human CD34 gene
Blood,
December 15, 2002;
100(13):
4410 - 4419.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Cooper, J. E. Bush, T. A. Fehniger, J. B. VanDeusen, R. E. Waite, Y. Liu, H. L. Aguila, and M. A. Caligiuri
In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells
Blood,
November 15, 2002;
100(10):
3633 - 3638.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. M. Fedorov, O. Yu. Tyrsin, T. Papadopoulos, G. Camarero, R. Gotz, and U. R. Rapp
Bcl-2 Determines Susceptibility to Induction of Lung Cancer by Oncogenic CRaf
Cancer Res.,
November 1, 2002;
62(21):
6297 - 6303.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Minagawa, H. Watanabe, C. Miyaji, K. Tomiyama, H. Shimura, A. Ito, M. Ito, J. Domen, I. L. Weissman, and K. Kawai
Enforced Expression of Bcl-2 Restores the Number of NK Cells, But Does Not Rescue the Impaired Development of NKT Cells or Intraepithelial Lymphocytes, in IL-2/IL-15 Receptor {beta}-Chain-Deficient Mice
J. Immunol.,
October 15, 2002;
169(8):
4153 - 4160.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-K. Ye, K. Maki, H.-C. Lee, A. Ito, K. Kawai, H. Suzuki, T. W. Mak, Y.-h. Chien, T. Honjo, and K. Ikuta
Differential Roles of Cytokine Receptors in the Development of Epidermal {gamma}{delta} T Cells
J. Immunol.,
August 15, 2001;
167(4):
1929 - 1934.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Alas, C. Emmanouilides, and B. Bonavida
Inhibition of Interleukin 10 by Rituximab Results in Down-Regulation of Bcl-2 and Sensitization of B-cell Non-Hodgkin's Lymphoma to Apoptosis
Clin. Cancer Res.,
March 1, 2001;
7(3):
709 - 723.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
T. Wekerle, J. Kurtz, M. H. Sayegh, H. Ito, A. D. Wells, S. Bensinger, J. Shaffer, L. A. Turka, and M. Sykes
Peripheral Deletion After Bone Marrow Transplantation with Costimulatory Blockade Has Features of Both Activation-Induced Cell Death and Passive Cell Death
J. Immunol.,
February 15, 2001;
166(4):
2311 - 2316.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kaisho, K. Takeda, T. Tsujimura, T. Kawai, F. Nomura, N. Terada, and S. Akira
I{{kappa}}B Kinase {{alpha}} Is Essential for Mature B Cell Development and Function
J. Exp. Med.,
February 12, 2001;
193(4):
417 - 426.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Domen and I. L. Weissman
Hematopoietic Stem Cells Need Two Signals to Prevent Apoptosis; BCL-2 Can Provide One of These, Kitl/c-Kit Signaling the Other
J. Exp. Med.,
December 11, 2000;
192(12):
1707 - 1718.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. R. Schmidt, B. Piekos, M. S. Cabatingan, and R. T. Woodland
Expression of a Human Coxsackie/Adenovirus Receptor Transgene Permits Adenovirus Infection of Primary Lymphocytes
J. Immunol.,
October 1, 2000;
165(7):
4112 - 4119.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Hakimelahi, H. R. Parker, A. J. Gilchrist, M. Barry, Z. Li, R. C. Bleackley, and M. Pasdar
Plakoglobin Regulates the Expression of the Anti-apoptotic Protein BCL-2
J. Biol. Chem.,
April 6, 2000;
275(15):
10905 - 10911.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Domen, S. H. Cheshier, and I. L. Weissman
The Role of Apoptosis in the Regulation of Hematopoietic Stem Cells: Overexpression of BCL-2 Increases Both Their Number and Repopulation Potential
J. Exp. Med.,
January 17, 2000;
191(2):
253 - 264.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Ogilvy, D. Metcalf, C. G. Print, M. L. Bath, A. W. Harris, and J. M. Adams
Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival
PNAS,
December 21, 1999;
96(26):
14943 - 14948.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Drouet, J. Mathieu, N. Grenier, E. Multon, J.-J. Sotto, and F. Herodin
The Reduction of In Vitro Radiation-Induced Fas-Related Apoptosis in CD34+ Progenitor Cells by SCF, FLT-3 Ligand, TPO, and IL-3 in Combination Resulted in CD34+ Cell Proliferation and Differentiation
Stem Cells,
September 1, 1999;
17(5):
273 - 285.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
K. AKASHI, M. KONDO, S. CHESHIER, J. SHIZURU, K. GANDY, J. DOMEN, R. MEBIUS, D. TRAVER, and I.L. WEISSMAN
Lymphoid Development from Stem Cells and the Common Lymphocyte Progenitors
Cold Spring Harb Symp Quant Biol,
January 1, 1999;
64(0):
1 - 12.
[Abstract]
[PDF]
|
|
|
|
|
|
|
J.M. ADAMS, D.C.S. HUANG, H. PUTHALAKATH, P. BOUILLET, G. VAIRO, K. MORIISHI, G. HAUSMANN, L. O'REILLY, K. NEWTON, S. OGILVY, et al.
Control of Apoptosis in Hematopoietic Cells by the Bcl-2 Family of Proteins
Cold Spring Harb Symp Quant Biol,
January 1, 1999;
64(0):
351 - 358.
[Abstract]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract
