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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3226-3239
Mcl-1 in Transgenic Mice Promotes Survival in a Spectrum of
Hematopoietic Cell Types and Immortalization in the Myeloid Lineage
By
Ping Zhou,
Liping Qian,
Christine K. Bieszczad,
Randolph Noelle,
Michael Binder,
Norman B. Levy, and
Ruth W. Craig
From the Departments of Pharmacology and Toxicology, Pathology,
Anatomy, and Immunology, Dartmouth Medical School, Hanover, NH.
 |
ABSTRACT |
Mcl-1 is a member of the Bcl-2 family that is expressed in early
monocyte differentiation and that can promote viability on transfection
into immature myeloid cells. However, the effects of Mcl-1 are
generally short lived compared with those of Bcl-2 and are not obvious
in some transfectants. To further explore the effects of this gene,
mice were produced that expressed Mcl-1 as a transgene in
hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate
viability enhancement in a wide range of hematopoietic cell types,
including lymphoid (B and T) as well as myeloid cells at both immature
and mature stages of differentiation. However, enhanced hematopoietic
capacity in transgenic bone marrow and spleen was not reflected in any
change in pool sizes in the peripheral blood. In addition, among
transgenic cells, mature T cells remained long lived compared with B
cells and macrophages could live longer than either of these.
Interestingly, when hematopoietic cells were maintained in tissue
culture in the presence of interleukin-3, Mcl-1 enhanced the
probability of outgrowth of continuously proliferating myeloid cell
lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo
homeostatic mechanisms controlling viable cell number, but these
constraints could be overridden under specific conditions in vitro.
Within the organism, Bcl-2 family members may act at "viability
gates" along the differentiation continuum, functioning as part of a
system for controlled hematopoietic cell amplification. Enforced
expression of even a moderate viability-promoting member of this family
such as Mcl-1, within a conducive intra- and extracellular environment
in isolation from normal homeostatic constraints, can substantially
increase the probability of cell immortalization.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE BCL-2 FAMILY contains a growing
number of members that promote cell viability, including Bcl-x, Mcl-1,
and A1. Mcl-1 was originally identified based on its rapid upregulation
in ML-1 human myeloblastic leukemia cells initiating monocytic
differentiation.1 Mcl-1 enhances viability on transfection
into murine myeloid progenitor (FDC-P1) and Chinese hamster ovary
cells.2,3 This enhancement is generally moderate in in
vitro systems, with viability being maintained for a shorter period of
time with Mcl-1 than with Bcl-23 and no enhancement being
observed in some cell lines.4 The effects of introduction
of Mcl-1 into an intact animal system have not yet been described.
The Bcl-2-related genes exhibit different tissue- and differentiation
stage-specific patterns of expression, which may be one reason for the
existence of a variety of family members with apparently similar
functions. In the case of Mcl-1, the pattern of expression in myeloid
cells in vivo is similar to that observed in the ML-1 cell line; that
is, expression is abundant at immature stages of differentiation and is
subsequently downregulated.5,6 Expression in lymphoid cells
likewise occurs at specific differentiation stages, with Mcl-1 being
abundant in lymph node germinal center cells that are undergoing
affinity maturation but scant in mature resting lymphocytes in the
mantle zone.6 This is the reciprocal of the pattern
observed with Bcl-2.6,7 Because of this differentiation stage specificity of expression, Mcl-1 is generally only detectable at
low levels in whole tissue extracts.6 Bcl-x and
A18 also display distinct patterns of expression during
differentiation. For example, Bcl-2 is predominant over Bcl-x at early
stages of lymphoid development, Bcl-x is predominant at intermediate
stages (eg, in double-positive thymocytes), and Bcl-2 is re-expressed and Bcl-x downregulated in mature cells (ie, in single-positive thymocytes9). Overall, various Bcl-2 family members are
differentially expressed in cells of different lineages at different
stages of development.
The effects of Bcl-2 family members have been studied in transgenic and
null (knock-out) mice, as well as in transfected cell lines. With Bcl-2
and Bcl-x, enhanced cell viability is observed in transgenic mice
whereas enhanced cell death occurs in null animals.9-16 The
null animals show differences between the different family members,
because Bcl-x-null mice exhibit embryonic lethality with apoptosis in
the hematolymphoid and nervous systems,17 whereas the
Bcl-2-null mice are normal at birth but subsequently undergo fulminent
lymphocyte apoptosis.18 Another difference is that Bcl-x
contributes to the survival of thymocytes at an intermediate
(double-positive) stage of differentiation, whereas Bcl-2 has effects
in more mature (single-positive) cells, mirroring the above-described
pattern of expression.16 Thus, in the intact animal, Bcl-2
family members exhibit developmental stage specificity of function as
well as of expression.
Several studies have suggested that Bcl-2 family members can promote
cell survival without necessarily affecting cell differentiation. This
was first observed when Bcl-2 was transfected into a hematopoietic cell
line that can undergo differentiation (FDC-PMix)19: When
transfectants were placed under apoptosis-inducing conditions, Bcl-2
promoted the survival of cells capable of differentiating normally.
Similar results were recently obtained when Bcl-2 was expressed as a
transgene in mice having a defect in the development of either the
lymphoid or the myeloid lineage. Mice lacking the interleukin-7 (IL-7)
receptor exhibit a decrease in T-cell precursors,20 which
is probably related to the fact that IL-7 normally promotes expression
of Bcl-2. The introduction of a Bcl-2 transgene allowed these cells to
survive and differentiate, resulting in a normalization of the T
lymphocyte pool.21,22 Analogously, Bcl-2 promoted the
survival and differentiation of monocytes in mice lacking monocyte
colony-stimulating factor.23 Finally, A1 has been reported to promote differentiation in addition to viability.24
Taken togther, these findings suggest a model in which Bcl-2 family members control viability at critical gates along the differentiation continuum, by either promoting cell viability and allowing continued differentiation, or, alternatively, allowing cell death.
Although Bcl-2 has been shown to enhance cell viability for several
days to weeks in a wide variety of cell lines, there have been only
isolated reports relating to cell immortalization. Here, whereas Bcl-2
alone has not been observed to allow immortalization, a combination of
EµBcl-2 plus Myc was found effective, allowing the outgrowth of
pre-B-cell lines from mouse bone marrow.25 Analogously,
mice transgenic for Bcl-2 alone initially exhibit lymphoproliferation,10 whereas mice transgenic for Bcl-2
plus Myc exhibit rapid tumorigenesis.26 Furthermore, a
fraction of mice transgenic for Bcl-2 alone develop tumors after a long
latency period, and these frequently exhibit alterations in the
endogenous c-myc gene.27 Finally, tumor cells expressing
both Bcl-2 and Myc die rapidly on explantation into tissue culture, but
can be propagated as immortal bipotential (lymphoid/myeloid) progenitor cell lines on a specific stromal layer.28 Thus, cell
immortalization in the presence of a combination of Bcl-2 plus Myc has
been observed under specific conditions.25,28
Because of the insights gained from studies of mice transgenic for
Bcl-2, we applied a transgenic approach to further our understanding of
Mcl-1, particularly of its effects in hematopoietic cells. We prepared
mice that prominently expressed the Mcl-1 transgene in hematolymphoid
tissues, using a mini-gene construct consisting of the Mcl-1 human
genomic locus and presumptive regulatory elements. The Mcl-1 transgene
caused viability enhancement in hematopoietic cells of various lineages
and at various stages of differentiation. As in transfected lines, this
effect was moderate and Mcl-1 seemed to act within the context of
endogenous homeostatic determinants of viability. Quite unexpectedly,
the transgene had a striking effect when myeloid cells were explanted
into tissue culture in the presence of IL-3. Here, we observed the
outgrowth of continuously proliferating cell lines (consisting of mast
cells or monocytes) each time transgenic cells were maintained in bulk
culture and from greater than 5% of clones that formed in semisolid
medium. In contrast, nontransgenic cells could be maintained in culture for a period of months, but were never observed to form a continuous cell line (immortalization frequency < 2.5 × 10-8),
in agreement with the results of previous
investigators.29-34 Taken together, our findings indicate
that enforced expression of even a moderate viability-enhancing member
of the Bcl-2 family such as Mcl-1 can contribute to a dramatic
alteration in cell fate: viability promotion by the Mcl-1 transgene, in
the context of a conducive intra- and extracellular milieu and in
isolation from homeostatic constraints, could substantially increase
the probability of escape from the normal limitation on cell lifespan to allow for immortalization.
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MATERIALS AND METHODS |
Preparation of the Mcl-1 transgene construct.
Cloned fragments of human genomic DNA were used to prepare a transgene
construct containing all exons and introns of Mcl-1, the
3 -untranslated region, and approximately 10.5 kb of the genomic 5-flanking region and 1.7 kb of the 3-flanking region
(Fig 1A). This was accomplished by ligation
of three genomic fragments from the two overlapping lambda phage clones
(clones B3 and B5). First, a 3.7-kb XbaI-XbaI fragment
(Fragment 1, Fig 1A) from clone B5 was ligated to a 2.8-kb
NotI-XbaI fragment (Fragment 2) from clone B3, and then
into the NotI-XbaI-digested pBluescriptSK+
vector. The resulting plasmid was then digested with NotI and SalI, releasing a fragment containing the above 6.5 kb of Mcl-1 along with, at its 3-end, 57 bp of vector multiple cloning site sequence terminating in a SalI site. This fragment was then
ligated to an 11-kb SalI-NotI fragment (Fragment 3)
from clone B3, and into SalI-digested pBluescript. A plasmid
containing the resultant 17.5-kb SalI-SalI insert
oriented correctly, pSS17.5, was purified on a cesium chloride
gradient,35 and the insert was purified by gel
electrophoresis/electroelution36 and Qiagen column
chromatography (Qiagen, Chatsworth, CA). The integrity of the Mcl-1
insert was confirmed by restriction enzyme mapping and DNA sequencing
of the ligation joints.
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| Fig 1.
The protein product of the Mcl-1 mini-transgene is
prominently expressed in hematolymphoid tissues. (A) The Mcl-1
mini-gene construct used in the generation of transgenic mice (pSS17.5
plasmid) contains all exons ( , including the 3-untranslated
region [ ] and introns ( ) of human Mcl-1. It also contains Mcl-1
5- and 3-genomic flanking regions ( ). Fragments 1, 2, and 3 are genomic subclones used in making the transgene construct.
Restriction sites are indicated as follows: S, SalI; X,
XhoI; B, BamHI; and Xb, XbaI. The BS3.0 genomic
subclone shown was used as probe to identify transgenic mice by
Southern blotting. (B) Expression of the Mcl-1 transgene was assayed in
a variety of tissues by Western blotting. ML-1 cells incubated with 5 × 10-10 mol/L TPA for 3 hours served as a positive
control. Each lane represents 50 µg of protein, except lane 13 which
represents 100 µg of protein. (C) Expression of the human Mcl-1
transgene in the spleen and lymph node (L.N.) of a transgenic mouse
(Tr.) was assayed in parallel with human lymph node. ML-1 cells
incubated with 5 × 10-10 mol/L TPA for 3 hours served as
a positive control. Each lane represents 100 µg of protein, except
lane 4 which represents 50 µg of protein. (D) Expression of the Mcl-1
transgene was assayed in enriched T- and B-cell populations from
transgenic mouse spleen. Unfractionated spleen from a transgenic (Tr.)
and a nontransgenic (Non-Tr.) mouse, as well as ML-1 cells incubated in
the presence or absence of 5 × 10-10 mol/L TPA for 3 hours served as controls. Each lane represents 50 µg of protein. (E)
Expression of the Mcl-1 transgene was assayed in cell lines derived
from cells from the spleen or bone marrow of transgenic mice. Lanes 1 through 3 (mast cell lines) represent 1 × 106 cells and
lane 4 represents 5 × 105 cells. Lanes 5 and 6 (monocytic
cell lines) and lane 7 represent 50 µg protein.
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Preparation of Mcl-1 transgenic mice.
Microinjection of insert DNA (4 ng/mL in 10 mmol/L Tris-Cl, pH 7.5, containing 0.25 mmol/L EDTA) was performed by DNX Corporation (Princeton, NJ). The founder mice (C57B6/SJL-F1) obtained were mated
with normal C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME). The
presence or absence of the Mcl-1 transgene was determined by Southern
blotting,35 using as a probe a subclone of human genomic
DNA (clone BS3.0 [in the Bluescript vector]) that had been labeled by
the random primer method.37 This probe detects a 16-kb
XhoI band in genomic DNA from mice made transgenic for human
Mcl-1 not in nontransgenic mice.
Expression of the Mcl-1 transgene.
Expression of the protein product of the human Mcl-1 transgene was
assayed by Western blotting using a rabbit polyclonal anti-Mcl-1 antibody that specifically detects human Mcl-1 and does not detect cross-reacting bands in mouse cells.5 Tissues were excised, minced rapidly, placed into tubes containing ice-cold tissue lysis buffer (50 mmol/L HEPES, pH 7.2; 150 mmol/L NaCl; 0.2% NP-40; 2 mmol/L
EGTA; 15 mmol/L EDTA; and 1% sodium dodecyl sulfate), homogenized with
a Tissue Tearor (Biospec Products, Inc, Racine, WI), boiled for 10 minutes, and centrifuged at 16,000g for 5 minutes (room
temperature). Protein concentration in the resultant supernatants was
assayed using the DC Protein Assay Kit (Bio-Rad, Hercules, CA).
Immunoblotting was performed using previously described
methods.2
T- and B-cell-enriched populations were separated as follows.
Enrichment for B cells was performed by complement-mediated lysis of T
cells.38 Briefly, spleen cells were obtained and erythrocytes removed using ACK lysis buffer (0.15 mol/L NH4Cl; 1.0 mmol/L KHCO3; 0.1 mmol/L EDTA, pH 7.2). Cells were then
incubated on ice for 45 minutes with an anti-Thy-1 monoclonal antibody
in Hank's balanced salt solution (HBSS; BioWhittaker, Walkersville, MD) containing 5% fetal calf serum (FCS). After collection by centrifugation, cells were incubated at 37°C for 30 minutes in HBSS
containing 5% FCS (HBSS/FCS) and 5% rabbit serum as a source of
complement. After three washes with cold HBSS/FCS, the T-cell-depleted population (generally 95% B cells) was assayed for cell viability and
protein concentration.
Enrichment for T cells was performed by depletion of B cells, which was
accomplished by panning on dishes coated with goat anti-mouse Ig
antibody. Briefly, after lysis of erythrocytes as above, spleen cells
were incubated at 4°C (with intermittent swirling) in plastic
dishes precoated with goat anti-mouse Ig, using a buffer of
phosphate-buffered saline ([PBS] 137 mmol/L NaCl; 2.7 mmol/L KCl; 10 mmol/L Na2HPO4; 1.8 mmol/L
KH2PO4, pH 7.4) containing 5% FCS. After 70 minutes, cells remaining in suspension were obtained, the panning
process repeated, cells in suspension again harvested, and the
B-cell-depleted population (generally approximately 90% T cells)
washed three times with HBSS/FCS and assayed for protein concentration.
Isolation and short-term in vitro culture of cells from spleen and
bone marrow.
Suspensions of spleen cells were prepared by dispersing freshly removed
spleen tissue in Delbecco's Modified Eagle medium containing 10%
fetal bovine serum (FBS), 50 U/mL penicillin, and 50 µg/mL
streptomycin (standard medium), and rinsing the splenocytes through a
210-µm Spectra/Mesh Polypropylene filter (Spectrum, Houston, TX).
After centrifugation (5 minutes at 200g), cells were
resuspended in standard medium, counted to determine the total number
of splenocytes recovered, and incubated at 4 × 106
cells/mL in tissue culture. Cell viability was monitored on subsequent days of culture by trypan blue dye exclusion.39 In some
experiments, the tissue culture medium was additionally supplemented
with 100 to 200 pmol/L IL-3 or IL-2 (R & D Systems, Minneapolis, MN).
Suspensions of bone marrow cells were obtained by flushing the tibia
with standard medium using a 10-mL syringe equipped with a 22-gauge
needle.
The fraction of cells of various lineages surviving on in vitro
culture, as compared with the number present on day 0, was calculated
as follows: (the number of viable T, B, or myeloid cells remaining on
day 1 or 3)/(the number of viable T, B, or myeloid cells initially
present on day 0). The numerator and denominator of this ratio were
calculated from the viable cell number (determined using trypan blue)
and the percentage of viable cells expressing lineage markers
(determined by flow cytometry as described below). Similarly, the
fraction of colony-forming cells surviving on in vitro culture was
calculated as the following ratio: (the number present on day 1)/(the
number present on day 0).
Flow cytometry.
The primary antibodies used were monoclonal rat anti-mouse antibodies
(fluorescein isothiocyanate [FITC]- or phycoerythrin [PE]-conjugated, or biotinylated forms) directed against the
following markers: CD3, CD5, and CD45R (B220) (Sigma Chemical Company,
St Louis, MO); CD4, CD8, IgM, CD34, CD13, Thy1.2, and Sca-1
(Pharmingen, San Diego, CA); CD11b, F4/80, c-kit, and Gran-1 (Caltag,
South San Francisco, CA); and MP12 and MP20 (Bachem, King of Prussia, PA). PE- or FITC-conjugated rat IgG was used as a negative control. Flow cytometry was performed using a FACSCAN (Becton Dickinson, San
Jose, CA) and a total of 10,000 cells (viable plus dead) was assayed
for each sample. When dead cells were present, the data were analyzed
by gating on the viable cell population with a reference set by the
propidium iodide staining. Where two-color staining was performed,
single-color staining was carried out in parallel to set the electronic
compensation.
Assay for IgE receptors was performed as follows: cells were incubated
for 45 minutes on ice with purified mouse IgE (Pharmingen; 1 µg/106 cells in 50 µL of staining buffer), washed with
staining buffer, and then incubated for 30 minutes on ice with an
FITC-conjugated rat anti-mouse IgE antibody (Pharmingen; 1 µg/106 cells in 50 µL of staining buffer). Cells were
washed and analyzed using a FACSCAN as above.
For each antibody used, the percentage positive cells was calculated as
follows: a threshold was set such that the fluorescence of at least
95% of the cells stained with the negative control antibody fell below
the threshold. Cells that exhibited fluorescence above this threshold
with the test antibody were considered positive.
Assay of colony formation in semisolid medium.
Cells obtained from the bone marrow were assayed for the ability to
form various types of colonies in methylcellulose medium. The medium
used for assay of myeloid/erythroid colonies was Methocult M3534
(Stemcell Technologies, Inc, Vancouver, Canada), which contains IL-3,
IL-6, stem cell factor, FBS, bovine serum albumin, 2-mercaptoethanol, insulin, and transferrin; this medium was supplemented with
erythropoietin (3 U/mL; R & D Systems). The medium used for assay of B
lymphoid colonies was Methocult M3630 (Stemcell Technologies), which
contains IL-7, FBS, and 2-mercaptoethanol. Bone marrow cells were
plated at 1.5 × 104 cells/35-mm dish for assay of
myeloid/erythroid colonies and at 4 × 104 cells/35-mm
dish for assay of B lymphoid colonies. Colonies were counted on day 7 using a Zeiss Axioskop inverted microscope (10× lens). In initial
tests, spleen cells were similarly tested for formation of myeloid
colonies, using the Methocult M3534 medium without erythropoeitin
supplementation. Spleen cells were plated at 1.5 × 105 cells/35-mm dish and the colonies that formed were
counted by eye on day 9.
Long-term in vitro culture for the derivation of cell lines from
spleen and bone marrow cells.
Cells from the spleen were incubated overnight in standard medium (5 × 105 cells/mL) and then transferred (at the same
cell density) into long-term culture medium which consisted of X-VIVO
15 medium (BioWhittaker) supplemented with 10% FBS, 200 pmol/L IL-3 (R
& D systems), and 20% WEHI-3B- conditioned medium prepared as
described previously.40 Cells from the bone marrow were
treated identically except that they were incubated in standard medium
for 4 days before transfer to long-term culture medium. Nonadherent and
adherent cells were maintained separately as follows. At weekly
intervals, nonadherent cells were collected by centrifugation and
recultured in fresh medium (5 × 105 cells/mL) in a
new flask. Remaining adherent cells were discarded for the first two
passages when fibroblasts were abundant. Thereafter, loosely adherent
monocytic cells were also observed. These cells were passaged by
reculturing loosely adherent cells tht were found in suspension at
confluence. Continued passage in this fashion resulted in decreasing
numbers of fibroblasts and the emergence of monocytic cell lines.
Tissue histology.
For light microscopy studies, fixation in 10% neutral buffered
formalin (Curtis-Matheson Scientific, Houston, TX); paraffin embedding;
sectioning and staining with hematoxylin and eosin; as well as
histochemical staining for myeloperoxidase, alpha naphthyl butyrate
esterase, and chloroacetate esterase were performed in the
Dartmouth-Hitchcock Hospital Pathology Department facility (Lebanon,
NH).
For transmission electron microscopy studies, samples were prepared as
follows: cells were rinsed in 0.1 mol/L phosphate buffer (pH 7.4)
containing 4% sucrose. They were then fixed in 2% glutaraldehyde/1% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 15 minutes
(room temperature), followed by replacement with fresh fixative and
fixation for an additional 1 hour (4°C). After fixation, cells were
rinsed in 0.1 mol/L phosphate buffer (pH 7.4) and postfixed in 1%
OsO4 in 0.1 mol/L phosphate buffer (pH 7.4). Cells were dehydrated through a graded series of ethanols and propylene oxide and
embedded in epon (LX112). Thin sections were stained with 2% uranyl
acetate in methanol for 20 minutes, followed by 5 minutes in Reynold's
lead citrate. Micrographs were taken at 80 kV on a JEOL 100CX electron
microscope (JOEL USA, Inc, Peabody, MA).
| |
RESULTS |
The Mcl-1 transgene is prominently expressed in hematolymphoid tissues.
Because Mcl-1 exerts effects in hematopoietic cells on
transfection,2 we wished to test for effects in
hematopoietic tissues in transgenic mice. To this end, we constructed a
minigene containing the Mcl-1 human genomic locus complete with
5- and 3-flanking regions (Fig 1A). We used this
construct, instead of lineage-specific expression constructs, in the
hopes of promoting transgene expression in a variety of hematolymphoid
tissues. This hope was based on the fact that a similar approach had
been used with other genes,41-44 where it was found to
result in elevated expression in tissues that normally express the gene
product. Thus, although data comparing the regulatory regions of human
and mouse Mcl-1 are not yet available, the approach of using human
genomic constructs for the preparation of transgenic mice has been
applied successfully for a variety of other genes. In addition, the
highly conserved carboxyl portion of the Bcl-2 that is important for
function is greater than 90% identical in the human and murine Mcl-1
gene products. Finally, the human gene product is known to have
viability-promoting effects in a murine hematopoietic cell
line.2 Using this approach, we obtained two transgenic
founder animals (female). These were mated with nontransgenic mice, and
offspring that were transgenic (heterozygous) for human Mcl-1 were
compared with nontransgenic littermates of the same sex, or, if not
available, nontransgenic animals of the same age and sex from
concurrent litters. The experiments described below were performed
using offspring from one of the founders, with similar effects being
observed with the other founder.
The mice obtained indeed exhibited prominent transgene expression in a
variety of hematopoietic and lymphoid tissues, establishing the use of
the approach involving a human genomic construct in transgenic mice.
Using an antibody that detects human (but not mouse) Mcl-1, the protein
product of the human transgene was found to be abundant in bone marrow,
lymph node, thymus, and spleen, and to be expressed at lower levels in
some other tissues (kidney, small intestine, uterus, lung, and liver;
Fig 1B). In the spleen, expression was prominent in both B- and
T-cell-enriched populations (Fig 1D). The level of Mcl-1 transgene
expression was considerably higher than the level of Mcl-1 expression
in human lymph node assayed in parallel (Fig 1C), and approached that
observed in ML-1 cells treated with 12-O-tetradecanoylphorbol
13-acetate (TPA) (Fig 1C and D). These levels are well
within the range known to produce viability-enhancing effects in
transgenic cell lines.2
The Mcl-1 transgene promotes survival in mature lymphoid and myeloid
cells but does not completely override endogenous determinants of cell
death.
Initial examination of the Mcl-1 transgenic mice showed enlargement of
the spleen, which was moderate but was observed in the majority of
animals (Fig 2A). Spleens were therefore
explanted from a series of 25 transgenic mice (6 weeks to 1 year in
age) to assay total splenocyte number. Twenty-three of these animals showed an increase in splenocyte number as compared with matched controls, the average increase being 1.6-fold (SE = 0.08; P < .01 Student's one-tailed t-test).
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| Fig 2.
The Mcl-1 transgene causes splenic enlargement and
enhances spleen cell survival in vitro. (A) Shown are spleens from two
12-week-old male littermates, one transgenic for Mcl-1 (left) and one
nontransgenic (right). (B) Spleens from transgenic and nontransgenic
mice were explanted, and the splenocytes were placed in suspension in
standard medium, incubated in tissue culture, and assayed daily for
cell viability by trypan blue dye exclusion. The points shown are the
mean ± SE for cells from 18 transgenic mice and 12 matched
nontransgenic controls. The significance of the difference between
nontransgenic and transgenic cells, as assessed by analysis of variance
with the Scheffé test, indicated a P value of <.01 for
days 1, 2, 3, and 4.
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In Bcl-2 transgenic mice, splenic enlargement is accompanied by
enhanced splenocyte survival on incubation in tissue culture. To assay
for such an effect in the case of Mcl-1, we monitored splenocyte
viability over 4 days of incubation in vitro. At every time point
assayed, viability was significantly higher in cultures from transgenic
animals than in parallel cultures from nontransgenic controls (Fig 2B).
Thus, the average percentage of viable cells declined to approximately
50% within 1 day in nontransgenic cultures, whereas an equivalent
decline required about twice as much time in transgenic cultures. The
enhancement of viability was particularly noticeable on day 4, when
nontransgenic cells had nearly all died whereas about 25% of
transgenic cells remained viable. In sum, Mcl-1 enhanced spleen cell
survival, a result that paralleled very closely previous findings in
transfected cell lines.2,3
We next monitored B- and T-cell markers in transgenic splenocytes, to
determine which lineage(s) were affected by Mcl-1. We found that the
increase in transgenic splenocyte number in vivo (Fig 2A) represented
an increase in both B and T cells, the relative proportions of these
two being unchanged (Fig 3A, day 0).
Likewise, the increase in transgenic splenocyte viability in vitro (Fig 2B) represented the survival of cells of both lineages (Fig 3A, day 3 where both B and T cells remain present). Overall, Mcl-1 promoted the
survival of cells of both lymphoid lineages, both in vivo and in vitro
(Figs 2 and 3A).
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| Fig 3.
The Mcl-1 transgene enhances the survival of mature
lymphoid (B and T) as well as myeloid cells but does not override
endogenous determinants of viability. (A) Spleen cells from transgenic
and nontransgenic mice were assayed by two-color flow cytometry for the
presence of B and T cell cell markers, either immediately on
explantation (day 0) or after 1 or 3 days of of incubation, as in Fig
2B. The dot plots shown were obtained by gating on the viable cell
population and the percentages of B220+ and
CD3+ cells, and cell-surface
IgM+ and CD5+ cells, represent the mean ± SD of two to three animals. Results from parallel assays are shown in
Table 1. The B/T cell ratio indicated was calculated from both sets of
markers. The number of cells recovered from the spleen in these
experiments averaged 2.9 ± 0.6 (SD) × 108 for
nontransgenic and 4.6 ± 1.3 × 108 for transgenic mice.
The percentage of viable cells on day 0 was greater than 95% and the
average percentage of viable cells on days 1, 2, and 3, respectively,
was 51% ± 6% (SD), 19% ± 4%, and 2.4% ± 0.7% for
nontransgenic mice and 75% ± 8%, 56% ± 5%, and 40% ± 8% for
transgenic mice. (B) The fractions of T lymphoid, B lymphoid, and
myeloid cells surviving on days 1 and 3 (compared with the initial
numbers present on day 0) were calculated from the experiment in (A).
The values for the T-cell markers CD3+ and
CD5+ cells were averaged, as were those for the B-cell
markers B220+ and IgM+; CD11b was assayed
in duplicate as a myeloid marker. Bars represent the SE of two to three
animals. On day 0, the total number of viable nontransgenic T, B, and
myeloid cells averaged 9.7 ± 0.1 × 107, 1.7 ± 0.2 × 108, and 1.5 ± 0.4 × 107 (SE of
three animals), and the total number of viable transgenic T, B, and
myeloid cells averaged 1.4 ± 0.1 × 108, 2.6 ± 0.3 × 108, and 2.8 ± 0.5 × 107. (C) Spleen cells
from Mcl-1 transgenic and nontransgenic mice were incubated in vitro as
in (A), with IL-2 or IL-3 (100 pmol/L) being added on day 1 as
indicated. On day 9, the viable cell population was assayed by flow
cytometry for CD11b and F4/80, CD11b and CD5, or B220. At this time,
the percentage of viable cells in transgenic cultures was 26% to 28%,
as determined by propidium iodide staining. Essentially no viable cells
remained in nontransgenic cultures to which no factor had been added on
day 1, and only small numbers of viable cells ( 1%) could be
detected in nontransgenic cultures to which growth factors had been
added. B, B220+ cells; T, CD5+ cells; M,
CD11b+ and/or F4/80+ cells. The
CD11b+ cells shown were CD5 , and the
CD5+ cells shown were CD11b, with 3%
of cells being CD11b+CD5+.
|
|
Histological examination agreed with the above flow cytometric results
in that the architecture of the spleen was generally preserved in
transgenic mice: splenic enlargement was not associated with any
dramatic follicular expansion, except in rare mice exhibiting expansion
of the while pulp. However, extramedullary hematopoiesis was very
active in the majority of transgenic animals. Occasional animals (two
of nine tested) also exhibited an increase in thymocyte number, but no
other consistent gross morphological alterations were noted.
Bcl-2 or Bcl-x transgenic mice made using lineage- or differentiation
stage-specific constructs often exhibit a preferential expansion of
one lymphoid lineage over the other and/or enhanced survival at
specific stages of differentiation (eg, the follicular expansion
observed in some strains of Bcl-2 transgenic
mice).11,16 However, the above histological and
flow cytometric observations did not suggest a preferential effect in
the Mcl-1 transgenic mice, which probably related to the fact that
Mcl-1 was expressed in both B and T cells (Fig 1C). To confirm a lack
of preferential effect and to further characterize the differentiation
phenotype of the transgenic splenocytes, we performed a more
comprehensive flow cytometric characterization. Lymphoid cells from
transgenic spleen were found to exhibit a normal mature cell phenotype,
and, other than the increase in spleen cell number, no overt
abnormality or preferentially expanded subpopulation was detected
(Table 1). Thus, B cells expressed
cell-surface IgM and IgD and T cells were predominantly either CD4 or
CD8 single positive. In sum, Mcl-1 seemed to cause enhanced survival in
various types of cells (Figs 2 and 3) without grossly altering the
pattern of cell differentiation or causing preferential expansion of a
particular cell type. The apparent contrast between these findings and
previous reports of the effects of lineage-specific Bcl-2 constructs is
probably more apparent than real, because Bcl-2 can promote survival in both lymphoid lineages and other cell types when appropriate promoters are used.
Whereas the proportions of B versus T cells in the spleen were not
altered in transgenic mice (Fig 3A, day 0), these proportions were
altered as cells were maintained in culture. B cells were initially
more abundant than T cells (B/T cell ratio of  1.8 on day 0, Fig 3A).
However, on day 1, nontransgenic cultures showed a substantial decrease
in the percentage of B cells and an increase in the percentage of T
cells (B/T cell ratio of 0.6), suggesting that B cells were dying more
rapidly than T cells. This decrease in the B/T cell ratio was delayed
in transgenic cultures. Calculation of the fraction of viable B cells
remaining on day 1 (as compared with day 0, Fig 3B) showed a value of
approximately 0.25 in nontransgenic cultures and 0.5 in transgenic
cultures; the fraction of viable T cells remaining at this time was
0.74 in both cases. In other words, the death of B cells occurred
earlier than that of T cells and this rapid B-cell death was partially
inhibited by Mcl-1. The slower process of T-cell death was also
inhibited by Mcl-1, because almost no nontransgenic T cells
remained present on day 3 but the fraction of remaining transgenic T
cells was greater than 0.6. The observation that the death of B cells
occurred more rapidly than that of T cells is in agreement with the
fact that mature, resting B cells (with the exception of memory cells)
are generally short lived compared with mature T
cells45,46. In sum, Mcl-1 enhanced cell survival in both
lymphoid lineages, and seemed to act within the context of other
determinants of viability such that T cells remained long lived
compared with B cells.
Myeloid cells, as represented by the CD11b marker, were present in low
numbers in the above splenocyte cultures (5% on average; Table 1).
These cells also exhibited extended survival in the presence of Mcl-1,
because the fraction of CD11b+ cells surviving on day 3 (compared with the initial number on day 0) was 0.07 in nontransgenic
and 0.25 in transgenic cultures (Fig 3B). By adding various growth
factors to the medium, we found that the survival of these cells could
be further extended by the addition of IL-3 (Fig 3C). Thus,
nontransgenic cells could not remain viable for an extended period even
in the presence of IL-2 or IL-3 (<1% viable on day 9), whereas a
proportion of the transgenic cells remained viable (26% to 28%). In
the absence of supplemental growth factors (or in the presence of
IL-2), the surviving transgenic cells consisted primarily of T cells.
However, in the presence of IL-3, the majority of surviving cells
(80%) expressed the general myeloid marker CD11b and/or the
monocyte/macrophage marker F4/80. In sum, like lymphoid cells,
transgenic myeloid cells exhibited enhanced survival in vitro, and this
was particularly pronounced in the presence of IL-3.
The Mcl-1 transgene enhances bone marrow hematopoietic capacity but
does not alter peripheral blood pools.
To extend the above observation of an effect in myeloid cells, we
cultured spleen cells in semisolid medium in the presence of factors
that promote the formation of myeloid colonies from precursors (IL-3,
IL-6, and stem cell factor). On average, 0.037% of transgenic spleen
cells formed macroscopically detectable colonies (SE=0.01%; n = 5),
which represented an approximate 3.2-fold increase (S.E. = 0.6) over
parallel nontransgenic cultures and accorded with the very active
extramedullary hematopoiesis noted on histological examination.
To assess more comprehensively the effects of Mcl-1 on hematopoietic
precursors, we obtained cells from bone marrow and assayed for the
formation of myeloid, erythroid, and lymphoid colonies in semisolid
medium containing the appropriate growth factors. When assayed
immediately on explantation of the spleen, transgenic animals showed a
doubling of the yield of myeloid and erythroid colonies and an
approximate fivefold increase in that of B lymphoid colonies
(Fig 4A). In addition, the colonies derived
from transgenic mice were larger in size than those from nontransgenic
controls (data not shown). We also tested for the ability of
colony-forming cells to survive in the absence of their specific growth
factors.47 This was performed by maintaining bone marrow
cells in liquid culture in standard medium before transfer to semisolid
medium containing the growth factors. Both myeloid and erythroid
colonies from transgenic mice showed enhanced survival (Fig 4B). In
sum, transgenic mice exhibited an increase in the number of
colony-forming cells that could be derived from transgenic bone marrow,
as well as an increase in survival when these cells were deprived of
specific growth factor requirements. These data showing enhanced
survival in a variety of hematopoietic precursors provide a parallel to the enhancement of survival observed in mature cells of various lineages.
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| Fig 4.
The Mcl-1 transgene enhances hematopoietic capacity in
the bone marrow although peripheral blood pools remain unaltered. (A)
Bone marrow cells from nontransgenic (Non-Tr.) and transgenic (Tr.)
mice were plated into methylcellulose medium containing factors
supporting the growth of either myeloid and erythroid precursors (left
set of bars) or B lymphoid precursors (right set of bars). The plating
density was 1.5 × 104 cells/35-mm dish for assay of
myeloid/erythroid colonies and 4 × 104 cells/35-mm dish
for assay of B lymphoid colonies. Colonies were counted after 7 days.
The values shown represent the mean ± SD of three independent
experiments using four transgenic and three control mice (P < .05 by Student's t-test). GM, colonies containing myeloid
(granulocyte-macrophage) cells; E, colonies containing erythroid cells;
GEMM, colonies containing both myeloid and erythroid
cells. (B) The bone marrow cells assayed in (A) were cultured in
standard medium (containing 10% FBS only) for 24 hours before plating
into growth factor-containing methylcellulose medium. The fraction of
colony-forming cells remaining on day 1 was calculated by comparison
with the number of present on day 0. The significance between
nontransgenic and transgenic animals, as determined by Student's
t-test, was P < .05 for both GM and lymphoid
colonies. (C) Bone marrow from transgenic and nontransgenic mice (10- to 12-weeks-old) was assayed by flow cytometry for the B220 and CD11b
cell-surface markers. The total number of cells recovered from the bone
marrow averaged 1.5 ± 0.3 (SD) × 107 cells for
nontransgenic mice and 1.3 ± 0.1 × 107 cells for the
transgenic mice. Bars represent the SE of three animals. The
CD11b+/B220+ ratio is listed on the figure.
The significance of the difference in this ratio in nontransgenic as
compared with transgenic animals, as assessed by analysis of variance
with the Scheffé test, which indicated a P value of
<.01. (D) The bone marrow cells assayed in (C) were also assayed for
the MP20 and MP12 markers. The percentage of
MP20+MP12+ double-positive cells averaged
11% ± 0.5% (SD) in nontransgenic and 14% ± 2% in transgenic
mice. The MP20+/MP12+ ratio is listed on
the figure. The significance of the difference in this ratio in
nontransgenic as compared with transgenic animals, as assessed by
analysis of variance with the Scheffé test, which indicated a
P value of <.05.
|
|
In view of the above alterations in precursor number, we surveyed the
cytometric marker profile of transgenic bone marrow. Transgenic mice
were found to exhibit a subtle increase in the proportion of
CD11b+ cells and decrease in the proportion of
B220+ cells, resulting in a 1.7-fold increase in the
CD11b+/B220+ ratio (Fig 4C). A small increase
in Gran1+ cells was also observed (Table 1). Because of the
subtle nature of these changes, we also used a second set of markers,
MP20 and MP1248, which delineate bone marrow
populations consisting primarily but not entirely of mature
myelomonocytic (MP20 single-positive) and lymphoid (MP12
single-positive) cells where erythroid cells are negative for both
these markers (MP12MP20). In the
Mcl-1 transgenic mice, we observed a shift in the proportions of these
markers that paralleled the shift in the
CD11b+/B220+ ratio (Fig 4D). These markers do
not represent populations identical to those marked by CD11b and
B220.48-51 In particular, the MP20 single-positive fraction
contains mature granulocytes and monocytes, whereas less mature cells
of the myeloid lineage reside in the double-positive
(MP20+MP12+) fraction, and (at even less mature
stages) in the MP12 single-positive fraction.48 The
proportion of MP20 single-positive cells was therefore smaller than the
proportion of CD11b+ cells, because CD11b is present on
some immature cells. Nonetheless, the results from these two different
sets of markers, when taken together, suggested that the presence of
Mcl-1 resulted in a shift in the proportion of myeloid relative to
lymphoid cells in the bone marrow.
Despite the above shift in the bone marrow, no change was apparent in
peripheral blood total or differential cell counts from transgenic mice
(approximately 107 red blood cells/mL and 6,000 to 7,000 white blood cells/mL of which 88% to 89% were lymphocytes, 10% were
segmented cells and 1% to 2% were monocytes). The above-described
moderate expansion of myeloid pools in the bone marrow was reminiscent
of the moderate expansion of lymphoid pools observed in the spleen (Fig
2). The fact that expansion in hematopoietic organs was not reflected in the peripheral blood suggests that the endogenous homeostatic mechanisms that regulate the ultimate numbers of viable cells in the
periphery are not affected by Mcl-1. We have recently obtained a
monoclonal antibody to Mcl-1 and are developing a flow cytometric assay
for the gene product. We plan to use this assay to determine whether
the transgene is expressed in periferal lymphoid and myeloid cells and
whether these cells exhibit enhanced survival. This will give an
indication of whether endogenous homeostatic mechanisms operate by
affecting transgene expression or alternatively by overriding the
effects of the transgene product.
The Mcl-1 transgene increases the probability of myeloid cell
immortalization on explantation to a conducive tissue culture
environment.
Because myeloid cells from transgenic mice displayed enhanced survival
in the presence of IL-3 (Fig 3C), we attempted to maintain these cells
in culture for an extended period of time. To our surprise, we found
that continuously proliferating cell lines consistently grew out from
bulk spleen-cell cultures from transgenic mice, a phenomenon that was
never observed in parallel nontransgenic cultures
(Fig 5A and
Table 2). Cell lines also arose from bone marrow cultures from transgenic (but not nontransgenic) mice (Fig 5B).
These cell lines consisted of immature mast cells as determined by
morphology, ultrastructure, and other characteristics such as the
presence of receptors for IgE (Fig 5D and E). Similar mast cells could
be maintained for some time in nontransgenic cultures, but invariably
eventually died (Fig 5 and Table 2) and never yielded a continuous cell
line. These cells resembled very closely the previously described "P
cells" which persist in cultures supplemented with IL-3 for up to 3 months, but eventually die without immortalization.29,32,33 Because we likewise did not observe immortalization in control cultures, the frequency of immortalization of nontransgenic cells could
only be estimated. An estimate would be that this frequency is less
than 2.5 × 10-8 because no immortalized cell lines
arose from a total of eight nontransgenic cultures initiated with 5 × 106 cells each (4 × 107 initial
cells; Table 2). In contrast to the results with nontransgenic cultures, a cell line arose each of nine times that nonadherent cells
from a transgenic mouse were maintained in the presence of IL-3 (Table
2). These cell lines expressed the Mcl-1 transgene (Fig 1E),
proliferated continuously provided IL-3 was present, and could be
maintained in culture for an indefinite period (>1 year and >70
population doublings).
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| Fig 5.
The Mcl-1 transgene promotes immortalization of monocytic
and mast cell lines. (A and B) Establishment of cell lines from
splenocytes and bone marrow cells from Mcl-1 transgenic mice. Cells
from a transgenic and a nontransgenic mouse were cultured in long-term
culture medium (containing IL-3 as described in Materials and Methods)
and assayed at approximately weekly intervals for the total number of
viable cells derived from the original culture, which contained a total
of 5 × 106 cells. (C) Light microscopic view of a
monocytic cell line (stained with Wright's Giemsa, original
magnification × 630). Histochemical staining showed this line to be
strongly positive for alpha-naphthyl butyrate esterase activity, weakly
positive for chloroacetate esterase activity, and negative for
myeloperoxidase activity. (D) Light microscopic view of a mast cell
line (stained with Wright's Giemsa, original magnification × 800).
(E) Electron microscopic view of a mast cell line (original
magnification × 8,300) showing characteristics typical of immature
mast cells, including large, often lobulated, nuclei and cytoplasmic
granules containing a central dense core and a mixture of particles and
vesicles.57,63 (F) Characterization of mast cell lines.
Cell surface markers and histochemical staining properties were assayed
for the indicated three cell lines. The percentage of
c-kit+ cells was high in all cell lines (99%), as was
the percentage of Sca1+ cells (89% for 4Q6BM, 90% for
3Y10BM, and 97% for 4Q6SP). Negligible percentages of cells expressed
CD11b, Thy 1.2, CD3, CD5, or B220. Histochemical staining for
chloroacetate esterase activity showed the following: 4Q6BM exhibited
staining in the majority of cells (varying intensity in different
cells); 3Y10BM contained a mixture of negative and weakly positive
cells; and 4Q6SP contained mostly negative cells, with a minority of
cells exhibiting weakly positive staining. The three cell lines were
negative for myeloperoxidase and alpha-naphthyl butyrate esterase
activities.
|
|
Whereas cell lines arose each time transgenic cells were maintained in
culture, Mcl-1 did not seem to cause direct immortalization of all of
the cells in the culture. Thus, during the initial 1 to 2 months in
culture, little or no net increase in cell number occurred (Fig 5A and
B); only after this "crisis"-like period did exponential growth
become apparent. In principle, a cell line can arise out of a single
immortalized cell. Therefore, to estimate the fraction of transgenic
cells subject to immortalization in the presence of Mcl-1, we plated
spleen cells in semisolid medium and picked 60 individual,
macroscopically visible clones from two separate transgenic mice. On
transfer to liquid culture, five of these clones were propagated
continuously. Nontransgenic clones did not develop into cell lines, as
expected from the above results with bulk cultures. The frequency of
immortalization of transgenic cells was then estimated to be in the
range of 3 × 10-5 (approximately 30 cells/106 transgenic spleen cells), based on the
above-described finding that approximately 0.037% of transgenic spleen
cells formed macroscopically visible colonies and that approximately
8% (5 of 60) of these were capable of immortalization. Given an
immortalization frequency for nontransgenic cells of less than 2.5 × 10-8, immortalization in Mcl-1 transgenic cultures
was estimated to be increased by a factor of greater than 1,000-fold.
In addition to the above mast cell lines obtained when nonadherent
transgenic cells were maintained in IL-3 (and adherent cells were
discarded), we observed monocytic cell lines to arise in three
experiments in which adherent cells were maintained separately in bulk
culture in the presence of IL-3 (nonadherent cells being discarded as
described in Materials and Methods; Fig 5C and Table 2). In contrast,
we did not observe cell lines to arise in the presence of IL-7. Thus,
beyond enhancing the survival of myeloid cells, Mcl-1 allowed for the
immortalization of mast and monocytic cells on incubation in the
presence of IL-3.
| |
DISCUSSION |
In the work described here, we prepared mice that expressed Mcl-1 as a
transgene in hematopoietic and lymphoid tissues. Initial observations
of these mice showed moderate splenic enlargement, which was associated
with increased numbers of B and T lymphocytes. The transgenic mice also
exhibited expansion of the myeloid relative to the lymphoid compartment
in the bone marrow. On explantation into tissue culture, mature B, T,
and myeloid cells, as well as immature colony-forming cells, showed
enhanced survival. The finding of lymphoid and myeloid expansion in the
spleen and bone marrow, respectively, could reflect the fact that much
lymphoid expansion normally occurs in the spleen whereas much myeloid
expansion normally occurs in the bone marrow. In other words, the Mcl-1
transgene amplified existing hematopoietic processes. The increased
yield of colony-forming cells, observed with both spleen and bone
marrow, probably also reflects the ability of Mcl-1 to amplify
hematopoiesis. Although viability enhancement by the Mcl-1 transgene
was observed in a spectrum of cell types at various stages of
differentiation, this effect was generally moderate (cell survival on
the order of several days to a week), mirroring previous results in
transfected cell lines.2,3 In addition, Mcl-1 did not
interfere with the ability of these cells to undergo differentiation,
paralleling similar observations with Bcl-2.19,52
The Mcl-1 transgene seemed to exert its effects within the context of
other homeostatic regulators that influence cell viability. For
example, whereas Mcl-1 enhanced the viability of a variety of
hematopoietic cells, T cells lived longer than B cells and cells
bearing macrophage markers could live longer than either of these. In a
similar vein, the enhanced viability observed in cells from the spleen
and bone marrow was not reflected in any change in peripheral blood
pools. These findings suggested that the final outcome, in terms of
viable cell number, depended on the combined action of Mcl-1 plus
additional endogenous factors, where such homeostatic factors were
capable of adjusting to the effects of the transgene. A similar
phenomenon has been observed with Bcl-2, where expression of the
transgene in T cells caused partial inhibition of negative selection in
the thymus although self-reactive cells did not reach the
periphery.12,53 Likewise, expression of the Bcl-2 transgene
in myeloid cells enhanced survival in vitro but did not increase pool
sizes in vivo.54 Overall, the effects of the Bcl-2 family
members in vivo are subject to normal homeostatic influences even under
conditions of enforced expression.
In addition to moderate survival-enhancing effects in a broad spectrum
of cell types, Mcl-1 had effects capable of resulting in a striking
alteration in cell fate when myeloid cells were cultured in the
presence of IL-3. The presence of the transgene resulted in an
increased probability of the outgrowth of continuously proliferating
cell lines. We first observed this phenomenon serendipitously, when
nonadherent spleen were maintained in the presence of IL-3. With bulk
nonadherent cell cultures, an immature mast cell line arose from each
transgenic culture, but not from the parallel nontransgenic control. By
picking individual clones from semisolid medium, we found that a
significant fraction of the transgenic cells that formed
macroscopically visible colonies could grow continuously as cell lines
(>5%). The overall frequency of immortalization was estimated to be
in the range of 3 × 10-5, in contrast to a frequency
of less than 2.5 × 10-8 in nontransgenic cultures.
The immature mast cells we observed are very similar to the P cells
initially characterized by Schrader et al.32,33 P cells are
immature mast cells that persist in tissue culture in the presence of
IL-3 (or IL-3 plus stem cell factor55-58), but survive for
a maximum of only several weeks or months.30,32 Whereas
cells persist for only a limited time when mouse hematopoietic cells
are cultured directly into IL-3-containing medium, continuous cell
lines do arise when cells are initially maintained in Dexter-type
long-term culture, where the cell inoculum is incubated for several
months in contact with a complex stromal layer.30,59 The
Mcl-1 transgenic cell lines arose without the use of a Dexter-type
long-term culture. Thus, Mcl-1 may have substituted for the effects of
the long-term culture environment. It will be interesting to determine
whether or not the Mcl-1 transgenic lines are tumorigenic, because
immortalized cells from Dexter-type cultures generally are not.
Overall, either the Mcl-1 transgene or the long-term culture
environment may provide a survival advantage that results in an
enhanced probability of cell immortalization.
In addition to the Mcl-1 transgene, several other factors may have
contributed to immortalization. These include the internal cell
environment, additional genetic changes, and the external cell milieu.
The importance of the internal cell environment is seen in the
observation that cell lines of myeloid but not lymphoid origin were
obtained with Mcl-1, contrasting with the pre-B or bipotential
progenitor lines previously observed with Bcl-2 plus Myc.25,28 It will be interesting to determine whether
immortalization with Mcl-1 is enhanced by oncogenes such as Myc, as is
the case with Bcl-2. The importance of additional genetic changes is
seen in the fact that immortalization in the presence of Mcl-1 did not
arise as an invariable response to the introduced gene. The importance
of the external cell milieu is seen in the fact that Mcl-1 transgenic
cell lines arose in the presence of IL-3 (but not IL-7) and in the
absence of endogenous homeostatic mechanisms that normally modulate
Bcl-2 family responses. The external environment (in this case, the
stromal layer) was similarly found to be critical in some cases of
immortalization with Bcl-2 plus Myc.28
Members of the Bcl-2 family normally act in specific cell types at
specific stages of differentiation. Indeed, one model for hematopoietic
cell amplification is that regulation may be exerted at a series of
viability gates present at critical points along the differentiation
continuum. These gates can be visualized as being under the control of
growth and differentiation factors, many of which regulate the
expression of Bcl-2 family members (eg, upregulation of Bcl-2 by
IL-7,21 A1 by
granulocyte-macrophage-CSF,60 Bcl-x by
CD40,15 and Mcl-1 by lymphocyte-conditioned
medium5). In other words, growth differentiation
factor-induced expression of viability-enhancing genes is postulated
to allow cells to pass through the gate and to continue to proliferate
and differentiate. A network of such gates would allow for a fine level
of control over cell amplification. This would also allow for rapid
changes in response to external stimuli, such as an increased or
decreased requirement for additional cells of a particular type.
Finally, the existence of successive gates would allow errors or
miscalculations from early gates to be corrected at subsequent gates.
Seen in the context of this model, the Mcl-1 transgene studied here
enhanced the ability of cells to pass through many of these viability
gates, but did not eliminate the self-correcting feature of the system as a whole. However, when cells expressing the Mcl-1 transgene were
removed from the endogenous constraints inherent in the system and
placed in a conducive growth factor-rich environment, self correction
was at least partially abrogated, allowing for eventual cell
immortalization and paralleling results with other
genes.61,62
In summary, Mcl-1 had moderate viability-enhancing effects in a
spectrum of hematopoietic cells in transgenic animals, but acted within
the context of the overall homeostatic regulation of viable cell
number. In addition, in specific types of myeloid cells, the presence
of Mcl-1 allowed for a high probability of immortalization when cells
were placed in a conducive environment in isolation from homeostatic
regulatory controls. This ability to have a long-term impact on cell
fate, shown under specific intra- and extracellular conditions, may be
more important in terms of diseases such as cancer than short-term
viability enhancement, which is observed with Bcl-2 family members in a
host of cell types and under a wide variety of conditions.
| |
FOOTNOTES |
Submitted March 31, 1998;
accepted June 29, 1998.
Supported by a grant from the National Cancer Institute (R01-CA57359).
The cloning of the Mcl-1 gene used in preparing the transgenic mice was
supported by R29-CA54385.
Address reprint request to Ruth W. Craig, PhD, Department of
Pharmacology and Toxicology, HB 7650, Dartmouth Medical School, Hanover, NH 03755.
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 Dr John Gearhart for expert advice in the early stages of this
project. We thank Drs Christopher Lowrey, Paul Wallace, and Nancy Speck
for their thoughtful reading of the manuscript. The transgenic founder
mice were prepared by DNX corporation. Electron microscopy was by
Louisa Howard and Charles Daghlian.
| |
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Abstract
Introduction
Abstract