Role of Bag-1 in the Survival and Proliferation of the Cytokine-Dependent Lymphocyte Lines, Ba/F3 and Nb2
Charles V. Clevenger,
Karen Thickman,
Winnie Ngo,
Wan-Pin Chang,
Shinichi Takayama and
John C. Reed
Department of Pathology and Laboratory Medicine (C.C.,
K.T., W.N., W.P.C.) University of Pennsylvania Medical Center
Philadelphia, Pennsylvania 19104
The Burnham Institute (S.T.,
J.R.) La Jolla, California 92037
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ABSTRACT
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The expression and function of the newly
identified Bcl-2- and Raf-1- binding protein, Bag-1, during the
cytokine-regulated growth of B and T cell lines was examined.
Immunoblot analysis of lysates from the interleukin-3 (IL-3)-dependent
B cell line Ba/F3, and the PRL-dependent T cell line Nb2, revealed that
variations in Bag-1 levels paralleled alterations in cellular
proliferation, viability, and apoptosis induced by the presence or
absence of growth factor. To test whether up-regulation of Bag-1 levels
altered cellular survival and proliferation, Ba/F3 cells were
transfected with a Bag-1 expression construct. The overexpression of
Bag-1 in transfected Ba/F3 cells induced an IL-3-independent state.
Such transfectants demonstrated sustained viability and proliferation,
with minimal apoptosis, in the complete absence of exogenous IL-3.
Bag-1 expression was also compared in glucocorticoid-sensitive Nb2
cells and a PRL-independent, glucocorticoid-resistant subline, SFJCD1,
during culture of these lines in dexamethasone (Dex). Bag-1 levels were
profoundly decreased by the addition of Dex to Nb2 cells, precedent to
the onset of apoptotic cell death. In contrast, Dex treatment or PRL
withdrawal had no effect on levels of Bag-1 within the SFJCD1 line.
These findings establish that the overexpression of Bag-1 in the
appropriate cellular context promotes cellular survival and growth,
events that may result from the juxtaposition of this protein with
mitogenic and antiapoptotic signaling pathways.
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INTRODUCTION
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The expansion of hematopoietic progenitors and effectors, mediated
by peptide growth factors, is required during an effective immune
response. This selective growth within the hematopoietic system occurs
as the summation of increased cellular proliferation and decreased
programmed cell death or apoptosis (1, 2, 3). Two peptide growth factors
that control such growth are interleukin-3 (IL-3) and PRL. IL-3 has
been found to increase the proliferation of most hematopoietic
precursors (4) and improve cellular survival by inhibiting apoptosis
(1, 5, 6). IL-3 acts as an immune stress factor, released by activated
T lymphocytes and mast cells, thereby mediating a paracrine
up-regulation of the pool of hematopoietic progenitors (4). Like IL-3,
PRL is also a peptide stress factor, whose secretion from the pituitary
and activated T cells is increased during an immune response. PRL is
necessary for both in vitro (7, 8, 9, 10) and in vivo
(11, 12, 13) lymphocyte proliferation. PRL is required for IL-2-driven T
cell proliferation; specifically, signals supplied from the PRL
receptor (PRLR) complex drive progression from G1 to S phase of the
cell cycle (14, 15). In addition, PRL has been found to inhibit
dexamethasone (Dex)-driven apoptosis of lymphocyte progenitors
(16, 17, 18). Thus, IL-3 and PRL may contribute to the overall
immmunoresponsiveness through their regulation of lymphocyte
apoptosis.
The regulatory role of apoptosis extends to many other aspects of the
immune response. The elimination of autoreactive lymphocyte precursors,
the lysis of target cells by cytotoxic T lymphocytes, and the loss of
CD4+ T cells during infection with human immunodeficiency virus are
mediated, in part, by apoptosis (19, 20). Although the intracellular
mechanisms that effect such cell death are incompletely characterized,
members of the Bcl-2 (for B cell lymphoma-2) family of genes are
thought to serve as central regulators of these phenomena (20, 21, 22).
Overexpression of Bcl-2 inhibits lymphocyte apoptosis initiated by
several disparate stimuli including the Fas ligand (23, 24, 25), T cell
cytolysis (26), glucocorticoids (27, 28, 29, 30), chemotherapy (31), and growth
factor withdrawal (32, 33, 34). Although the linkages between Bcl-2 and the
downstream factors that directly effect apoptosis are incompletely
characterized (35), Bcl-2 function is significantly modulated by
heterodimerization with other Bcl-2 family members (36, 37, 38). For
example, the extent of Bcl-2 and Bax homo- and heterodimerization is
thought to contribute to the control of cellular survival. In addition
to Bcl-2 family members, other proteins, most notably Bag-1, have been
found to interact with Bcl-2 and modulate its function. Bag-1 (for
Bcl-2-associated anti-death gene 1) was identified by the screening of
a murine embryo cDNA library with recombinant Bcl-2 (39). The
significance of the in vitro and in vivo
interaction of Bag-1 with Bcl-2 was confirmed by overexpression of
Bag-1 in Jurkat and NIH3T3 cells. Overexpression of Bag-1 promoted the
survival of these transfectants to a variety of apoptotic stimuli.
Thus, the multimeric interactions of the Bcl-2/Bag-1/Bax complex, as
regulated by the stoichiometry of its constituents, may significantly
affect cellular survival.
Despite the linkage between Bcl-2 family overexpression and apoptosis,
little is known of the regulation of these gene products in
nontransfected cell lines experiencing stimuli that trigger cell death.
If the regulation of such protein levels is central to the control of
apoptosis, such changes, at the protein level, should be detectable by
biochemical analysis either preceding and/or during entry into the
apoptotic pathway. In this study, we tested whether the expression of
the Bcl-2-binding protein, Bag-1, was associated with apoptosis induced
by growth factor withdrawal and/or dexamethasone treatment of
cytokine-dependent lymphocyte lines Ba/F3 and Nb2. These data revealed
a direct association between Bag-1 levels and cellular growth. To
confirm that increased levels of Bag-1 could enhance cellular
proliferation and viability during growth factor deprivation, a Bag-1
expression construct was transfected into Ba/F3. Examination of several
independently obtained transfectants has revealed that overexpression
of Bag-1 confers growth factor independence to the Ba/F3 line.
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RESULTS
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Bag-1 Expression Is Regulated during IL-3-Driven Ba/F3
Proliferation and Apoptosis
To determine whether an association existed between the levels of
the Bcl-2-binding protein, Bag-1, and apoptosis induced by cytokine
withdrawal, immunoblot analysis was performed on lysates obtained from
the lymphocyte lines, Ba/F3 and Nb2. Significant decreases in Bag-1
levels were noted in both cell lines during withdrawal of IL-3 and PRL,
respectively. To expand upon these preliminary findings, the dependence
of Ba/F3 on IL-3 for cell growth (Fig. 1A
) and viability
(Fig. 1B
) was correlated with Bag-1 expression with respect to dose and
time (Fig. 1
, C and D). Ba/F3 cells cultured in the absence of IL-3
expressed 6- to 8-fold less Bag-1 than parallel cultures incubated in
the presence of IL-3. This was due to a modest (50%) increase in Bag-1
levels in the IL-3-stimulated cultures and a 4-fold decrease in Bag-1
levels in IL-3-deprived cultures. As described below, the loss of
cellular viability, as measured by trypan blue uptake, was found to
occur shortly after the apoptosis-associated phenomena of cleavage and
loss of cellular DNA (Fig. 3
). The temporal decreases in Bag-1
expression were noted to occur in rough approximation with the
initiation of endonucleolytic DNA cleavage. Taken together, these data
indicate an association between decreased Bag-1 expression and the
onset of apoptosis.

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Figure 1. Association between Bag-1 Expression and
IL-3-Dependent Growth and Survival of the Murine B Cell Lymphoma Ba/F3
Ba/F3 cells were cultured in the presence of varying concentrations of
murine IL-3; at various time intervals, cell growth (A) and survival
(B) were measured using trypan blue exclusion. C, Parallel measurement
of Bag-1 levels in IL-3-stimulated Ba/F3 were performed by immunoblot
analysis using an anti-Bag-1 antiserum. Probing of a duplicate blot
with preimmune serum demonstrated the specificity of these results
(data not shown). Molecular mass standards indicated at
left are in kilodaltons. Bag-1 levels
below blot represent densitometric quantification of the
Bag-1 protein normalized to total cellular protein. D, Temporal
decrease of Bag-1 levels during IL-3 deprivation as measured by
scanning densitometry of anti-Bag-1 immunoblots. As in Fig. 1C , these
values in arbitrary units (au) were normalized for total cellular
protein.
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Figure 3. The Dependence of Ba/F3 Cells on IL-3 for Cell
Growth and Viability Is Abrogated by the Transfection of Bag-1
Parental and transfected Ba/F3 cells were cultured at various
times in the absence of IL-3. Cell growth (A) and viability (B) were
assessed as per Fig. 1 . Quantitative assessment of apoptosis during
IL-3 deprivation was performed by two independent assays. C, Cells
with endonucleolytic cleavage were labeled with ddUTP in the presence
(or absence as control) of TdT; the incorporated ddUTP was subsequently
detected with a fluorescein-conjugated anti-digoxigenin antibody.
Incorporation of this fluorescent marker of DNA cleavage was then
assessed by flow cytometry. D, Loss of cellular DNA, as manifested by a
hypodiploid DNA content, was assessed by flow cytometric analysis of
propidium iodide-stained cells.
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Overexpression of Bag-1 Permits Sustained Ba/F3 Viability and
Proliferation in the Absence of IL-3
To explore whether up-regulation of Bag-1 levels could inhibit
cellular death induced by cytokine withdrawal, stable transfectants of
Ba/F3 that constitutively expressed Bag-1 were generated. High levels
of Bag-1 were observed in clones containing the Bag-1 expression
construct (Fig. 2A
), in comparison to either the
parental line or control transfectants that received the same vector
lacking Bag-1. Similar to the parental Ba/F3, control transfectants
demonstrated significant decreases in Bag-1 levels as a function of
IL-3 deprivation (Fig. 2B
). When IL-3 was withdrawn from these
subclones, sustained proliferation and viability were observed in only
those transfectants that overexpressed Bag-1 (Fig. 3
, A
and B). Indeed, the viability of the Bag-1 transfectants in
IL-3-deficient medium paralleled that of the parental line raised in
medium containing IL-3 (see Fig. 1B
). Concurrent with the enhanced
survival, the Bag-1 transfectants continued to proliferate in the
absence of IL-3 (Fig. 3A
); however, a cell density of approximately
2-fold greater than the parental line was reached (see Fig. 1A
).
Additional studies revealed that if the IL-3-deficient medium was not
changed between 4860 h after culture initiation, the Bag-1
transfectants would demonstrate a decrease in viability and
proliferation. However, thrice-weekly refeeding with medium lacking
IL-3 permitted continued growth and viability (>95%) for at least 2
months.

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Figure 2. Expression of Bag-1 in Parental and Transfected
Ba/F3 as a Function of IL-3 Deprivation
Ba/F3 cells were stably transfected with Bag1-pCMV or Neo-pCMV
and cultured for various times without IL-3 as indicated (A). Cell
lysates from these cultures were prepared and subjected to sequential
SDS-PAGE and immunoblot analysis using an anti-Bag-1 antiserum. Bag-1
levels in both the Ba/F3 parent and control Neo-pCMV transfectants
decreased temporally during IL-3 deprivation (B). Levels of Bag-1 were
determined by immunoblot analysis of cell lysates using an
anti-Bag-1 antibody as above; the control blot represents parallel blot
probed with preimmune serum. Results similar to the control clone
Neo-pCMV-1 were obtained for Neo-pCMV-3 (data not shown).
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To confirm that the improvement in cell viability of the Bag-1
transfectants was secondary to a decrease in the percentage of cells
undergoing apoptosis, three independent measures of programmed cell
death were performed. Two of these measures, presented in Fig. 3
, C and
D, were flow cytometry-based assays that respectively measured the
percentage of cells undergoing endonucleolytic DNA cleavage [as
measured by the incorporation of digoxigenin labeled dUTP (ddUTP) by
terminal deoxynucleotide transferase (TdT)] or DNA degradation (as
measured by hypodiploid DNA content of propidium iodide-stained cells).
These analyses demonstrated that, in contrast to the transfected
controls and the parental line, the Bag-1 transfectants had a
significant reduction in the percentage of cells undergoing apoptosis
as a result of IL-3 withdrawal. Further analysis of these DNA content
histograms revealed an increase in the %S-phase fraction of the Bag-1
transfectants by approximately 2-fold (data not shown). Taken together,
these data indicate that constitutive overexpression of Bag-1 in the
Ba/F3 cell line induces an immortalized, IL-3-independent state that
prevents apoptosis upon withdrawal of IL-3 and permits continued
proliferation under such conditions.
Effects of PRL and Dex on Nb2 Cell Growth Viability and
Apoptosis
To extend the data obtained with the Ba/F3 cells, the
growth, viability, and apoptosis of PRL and Dex-treated Nb2 cells were
ascertained (Fig. 4
) at concentrations of PRL (5 ng/ml,
0.2 nM) and Dex (100 nM) previously determined
to induce maximal proliferation or apoptosis (16, 17), respectively
(Fig. 4
, A and B) The addition of Dex to Nb2 cells in the absence of
exogenous PRL was found to inhibit proliferation and induce a profound
decrease in viable cell number. The extent of Dex-induced apoptosis in
Nb2 cells was examined with the same methodology used for Ba/F3 (Fig. 4
, C and D) and confirmed that a significant increase in apoptosis
(also confirmed morphologically) occurred in Dex-treated cultures.

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Figure 4. Effects of PRL and Dex on Rat T Cell Lymphoma Nb2
Growth and Survival
Cell growth (A) and viability (B) of Nb2 cells were measured at various
times in the presence or absence of 5 ng PRL/ml or 100 nM
Dex, as per Fig. 1 . Apoptosis was assessed by flow cytometric
measurement of endonucleolytic cleavage (C) or loss of cellular DNA
content (D), as per Fig. 3 .
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In contrast to Dex-treated Nb2 cultures, the viable cell number
of Nb2 cells cultured in the presence of both exogenous PRL and Dex was
relatively stationary. Concomitantly, the incidence of apoptotic death
within these cultures was less marked than in cultures that received
Dex only. Of note, parallel growth and viability curves were observed
between cultures that received both or neither of the two hormones.
The Regulated Expression of Bag-1 Found in Nb2 Is Absent in the
PRL-Independent Nb2 Subline, SFJCD1
The expression of Bag-1 in Nb2 cells was measured by
immunoblot analysis (Fig. 5
). For these experiments, Nb2
cells were deprived of PRL for 24 h and then restimulated with
various concentrations of PRL or Dex before cell lysates were prepared
at various time intervals and immunoblot analysis of Bag-1 was
performed. The regulation of Bag-1 expression in Nb2 cells by PRL was
dose-dependent (Fig. 5A
), whereas a 5-fold decrease in Bag-1 levels was
observed in PRL-deficient cultures. In contrast, an inverse
relationship between Bag-1 levels and Dex concentration was noted (Fig. 5B
), with an approximately 15-fold decrease in Bag-1 levels in Nb2
cultures treated with 13 µM Dex. Further delineation of
the temporal regulation of Bag-1 levels in response to concomitant PRL
(5 ng/ml) and/or Dex (100 nM) stimulation is presented in
Fig. 5C
. These data revealed that treatment with only Dex induced
a rapid decline (after 6 h) in Bag-1, with 10-fold reduction in
levels by 24 h. Dex also induced decreases in Bag-1 protein levels
in PRL-stimulated Nb2 cells, but the effects were less striking than in
cultures that received Dex alone. Taken together, these data indicate
that Bag-1 levels correlate with cell growth and apoptosis in Nb2 cells
during treatment with Dex, PRL, or a combination of these reagents.

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Figure 5. The Regulated Expression of Bag-1 in Nb2 Cells Is
Absent in the Nb2-Derived, PRL-Independent SFJCD1 Subclone
Measurement of Bag-1 levels by immunoblot analysis in Nb2 cells
stimulated with PRL as a function of dose and time (A). The
concentrations of PRL used were either submitogenic (0.05 ng/ml), or
induced half-maximal (0.5 ng/ml) or maximal (5 ng/ml) growth. Bag-1
levels below blot represent densitometric quantitation
of the Bag-1 protein normalized to total cellular protein. B,
Measurement of Bag-1 levels by immunoblot analysis of lysates from Nb2
cells treated with Dex as a function of dose. Reported Bag-1 levels
were quantitated as per Fig. 5B . C, Temporal measurement of Bag-1
levels in lysates from Nb2 cells treated with 5 ng PRL/ml and/or 100
nM Dex as determined by immunoblot analysis with an
anti-Bag-1 antiserum. The relative Bag-1 levels reported here are
normalized to total cellular protein. D, Bag-1 expression in Nb2 or
SFJCD1 cells treated with 100 nM Dex or PRL-deprived for
48 h.
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To extend these correlations further, Bag-1 levels were examined
in the PRL-independent SFJCD1 T-cell line during PRL withdrawal or Dex
treatment. Unlike Nb2, this PRL-independent subline is resistant to
Dex-induced apoptosis (40, 41). As seen in Fig. 5D
, PRL withdrawal or
treatment with Dex (100 nM) had little effect on Bag-1
levels or on the viability of the SFJCD1 subline, in contrast to the
parental Nb2 line. These data, therefore, further confirm an
association between Bag-1 levels and Nb2 growth and survival,
suggesting a regulatory role for this protein in Nb2 cells, similar to
that seen in Ba/F3.
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DISCUSSION
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The receptors for IL-3 and PRL belong to the growth
factor/cytokine receptor superfamily that is widely expressed within
the hematopoietic system (42, 43, 44). Ligand stimulation of progenitor
cells promotes proliferation, survival, and differentiation of select
cell lineages, resulting in a coordinated immune response. A common
structure/function aspect of the growth factor receptor superfamily is
its lack of intrinsic kinase activity (42). Thus, signaling via these
receptors is mediated by associated transduction factors. Signals
supplied by receptor-associated kinase cascades, such as the JAK/STAT
and the Ras/Raf pathways (45, 46, 47, 48, 49, 50), are believed in part to transmit
signals from this receptor superfamily that initiate cell cycle
progression. In contrast, the structure/function basis for the
immediate antiapoptotic signals supplied by these receptors is
incompletely understood. Nevertheless, alterations in the levels of the
Bcl-2 family, induced by overexpression of transfected gene constructs
(i.e. Bcl-2 and Bax), clearly alters the survival, but not
the proliferation, of cytokine-dependent cell lines (33, 36, 51).
Overexpression of the Bcl-2 gene in transgenic animals also inhibits
the in vivo apoptosis of select lymphocyte progenitor
populations (27, 52, 53). Taken together, these data suggest that
alterations in the levels of members of the Bcl-2 family may control
apoptosis by several stimuli, including growth factor/cytokine
withdrawal. Initial studies performed in our laboratory, however,
revealed no significant changes in the levels of Bcl-2, Bax, or
Bcl-XL proteins during stimulation with or withdrawal of
growth factor from the Ba/F3 or Nb2 lines (not shown). These
observations largely agree with a previous report (54), which
demonstrated modest changes in the levels of Bcl-2 and Bax protein
during stimulation of Nb2 with PRL, despite the significant induction
of Bcl-2 and Bax mRNA during such treatment.
In contrast to Bcl-2, Bax, and Bcl-XL, these studies
indicate that the level of the Bcl-2-associated protein, Bag-1, was
closely associated with the growth (i.e. the proliferation
and inhibition of apoptosis) of both the Ba/F3 and Nb2 lymphocyte
lines. Coexpression of Bag-1 and Bcl-2 in Jurkat T cells resulted in a
synergistic improvement in cellular viability secondary to apoptotic
stimuli induced by Fas, staurosporine, or cytotoxic T lymphocytes (39).
These effects were not noted in Jurkat transfectants that overexpressed
Bag-1 alone. In contrast, the overexpression of Bag-1 alone in 3T3
fibroblasts was sufficient to inhibit staurosporine-induced apoptosis.
These data suggest that the function of Bag-1 is influenced by its
cellular environment and the nature of the apoptotic challenge. This
hypothesis was further confirmed in the present study, which
demonstrated that overexpression of the Bag-1 in Ba/F3 resulted in the
induction of IL-3 independence. Thus, in the absence of IL-3, Bag-1
transfectants maintained high viability and continued to proliferate at
a rate that exceeded that of the IL-3-stimulated parental line. Growth
factor independence has been conferred on IL-3-dependent lines
previously by the cotransfection of Bcl-2 and Myc (51), an effect not
observed when either of these gene products was individually
transfected (33, 34). Thus, these data suggest that the functional
effects of Bag-1 may extend beyond the downstream effectors of Bcl-2
action.
The ability of Bag-1 to induce factor-independent cell growth may
be related to its interaction with signaling factors intimately
associated with apoptosis and mitogenesis, namely Bcl-2 and Raf-1. As
part of a signaling cascade widely used by the growth factor receptor
superfamily, Raf is a central serine/threonine kinase that controls
cell cycle-dependent gene expression via a kinase cascade that induces
the phosphorylation of specific transcription factors (i.e.
Myc, Jun, and p62TCF) (55). These transcription factors are
substrates for mitogen-activated protein kinase (56), which in turn is
a substrate for mitogen-activated kinase kinase (MEK), the only
high-affinity substrate of Raf-1 known to date (57, 58, 59). Raf-1 activity
is allosterically modulated by the signaling proteins Ras, 143-3, and
as most recently identified, Bag-1 (60, 61, 62, 63, 64). Like 143-3, Bag-1
binding occurs in the carboxy terminus of Raf-1 (60). Although this
binding site represents the kinase domain of Raf-1, significant
phosphorylation of Bag-1 does not occur. In vitro and
in vivo data indicate, however, that this interaction
up-regulates Raf-1 kinase activity (60). Recent studies indicate that
Bcl-2 and Raf-1 can be coimmunoprecipitated (32, 65, 66) and that the
conserved BH4 domain within Bcl-2 is required for interaction with both
Raf-1 and Bag-1. Taken together, these data suggest that the
Bcl-2/Raf-1 interaction may be facilitated by Bag-1. It remains to be
determined whether the Bcl-2/Raf-1/Bag-1 complex is targeted to
intracellular membranes where Bcl-2 resides (67), such as the
mitochondrial outer membranes, nuclear membrane, endoplasmic reticulum,
etc. Furthermore, the stoichiometric relationships and functional
significance of each member of the Bax/Bcl-2/Bag-1/Raf-1 vis-a-vis
apoptosis requires further elaboration. It is interesting to note that
the overexpression of Raf-1 in growth factor-dependent cells can induce
a spectrum of changes related to cell type, ranging from growth factor
independence to apoptosis (65, 66). Thus, alterations in Bag-1 levels
may directly effect the coupling and composition of the
Bcl-2/Bag-1/Raf-1 complex, regulating its function during cellular
proliferation and apoptosis. Decreases in Bag-1 levels (as seen in
growth factor-deprived cells), therefore, may lead to an uncoupling of
the complex and decreased viable cell growth. Conversely, the increased
levels of Bag-1 may support the association of this signaling complex
and stimulate the increased proliferative rates and cell densities
observed in the Bag-1 transfectants. Alternatively, increases in Bag-1
levels may lessen the dependence on other rate-limiting nutrients or
growth factors within the medium, thereby enabling enhanced rates of
growth.
Like the IL-3-dependent Ba/F3 B-cell line, the PRL-dependent Nb2 T cell
line has served as an excellent model for examining growth factor
signal transduction. Previous studies have shown that treatment of this
line with PRL or Dex results in mitogenesis or apoptosis, respectively
(16, 17, 40). Addition of both hormones simultaneously to Nb2 cells
inhibited proliferation and diminished, but did not entirely prevent,
apoptosis. These in vitro data support several lines of
in vivo research, indicating that one mechanism through
which the body may regulate its immunoresponsiveness during periods of
stress is by alterations in the PRL/Dex balance (11, 12, 13, 68, 69, 70). As
evidenced by the SFJCD1 line, escape from these regulatory mechanisms
can impart growth factor independence (17). The SFJCD1 subline was
derived from Nb2; karyotypic analysis has demonstrated that a de
novo translocation involving chromosomes 14 and 17 occurred within
the SFJCD1 cells (41). Significant differences in PRLR-associated
signaling also exist between these PRL-dependent and independent lines.
During PRL stimulation of Nb2 cells, the activation of a cascade
involving guanine nucleotide exchange factors (Sos- and Vav-associated
activity), Ras, Raf, and mitogen-activated protein kinase has been
documented (71, 72, 73). In contrast, both the PRL-independent Sp (71) and
SFJCD1 cell lines contain a constitutively activated Raf-1,
irrespective of the presence of PRL. Thus, these data indicate that
some alteration in the inducible PRLR-associated kinase cascade has
occurred in SFJCD1 cells. Although many possibilities exist, this
up-regulation of Raf-1 kinase activity may result from its allosteric
activation secondary to the constitutively elevated levels of Bag-1
found in SFJCD1. Studies to evaluate this hypothesis are currently
underway in our laboratory. Nevertheless, these data, coupled with
those from the Ba/F3 system, suggest that Bag-1 is involved in the
integration of the mitogenic and apoptotic signaling cascades within
the hematopoietic and the immune systems.
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MATERIALS AND METHODS
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Cell Culture
The mouse IL-3-dependent pro-B cell line, Ba/F3 (74), was
maintained in RPMI 1640 medium supplemented with 10% FBS and 1%
penicillin/streptomycin in the presence of 1 ng IL-3/ml (Prepro-Tech,
Rocky Hill, NJ). For experimental purposes this medium was used with
varying concentrations of IL-3. The rat pre T cell lymphoma line Nb2/11
(hereafter referred to as Nb2) and the SFJCD1 cell lines (both obtained
from Dr. Peter Gout) were cultured in Fishers medium containing 10%
FBS, 10% gelding horse serum, 10-4 M
ß-mercaptoethanol, and penicillin/streptomycin (40, 71). For
experimental purposes, both the Nb2 and SFJCD1 cells were stimulated
with varying concentrations of PRL (NIDDK) and Dex (Sigma, St. Louis,
MO) in a chemically defined medium consisting of DMEM supplemented with
sodium selenite, linoleic acid, insulin, transferrin (ITS+ supplement,
Becton-Dickinson, San Jose, CA), ß-mercaptoethanol, and
penicillin/streptomycin. For some studies, 5 ng PRL/ml and/or 100
nM Dex were used; these concentrations, respectively, have
been found to induce maximal cell proliferation (71) or apoptosis (16).
The rates of cell death induced by cytokine withdrawal for Ba/F3 and
Nb2 differ and have been well documented (40, 71, 74); loss of
exogenous IL-3 from the culture medium of Ba/F3 induces 90% cell death
by 2440 h, while removal of PRL from the Nb2 medium induces a similar
level of death by 4060 h. Cell density and viability were assessed by
cell labeling with trypan blue; all measurements were performed in
triplicate by hemocytometry on at least 200 cells per point.
Apoptosis Measurements
Cell apoptosis was measured by three independent methodologies,
as follows: 1) Endonucleolytic DNA cleavage was detected in the Ba/F3
and Nb2 using a commercially available kit (Apoptag, Oncor,
Gaithersburg, MD), as described previously (75). Briefly, 2 x
106 washed cells were sequentially fixed in 2%
paraformaldehyde and 70% ethanol; DNA cleavage was then detected
through the incorporation of ddUTP by terminal deoxynucleotide
transferase. To determine the background level of ddUTP incorporation,
parallel samples were incubated in the absence of terminal
deoxynucleotidyl transferase as a control. Incorporated ddUTP was then
subsequently labeled with a fluorescein-conjugated antidigoxigenin
antibody. 2) Loss of cellular DNA, as manifest by a hypodiploid DNA
content, was measured a described previously (76). Briefly, 2 x
106 washed cells were fixed in 70% ethanol, treated for 20
min with 150 units RNAseA (Worthington Biochemical Corp., Freehold,
NJ)/ml, and labeled with 50 µg propidium iodide (Calbiochem, San
Diego, CA)/ml. 3) Alteration in cellular morphology, as manifest by
chromatin condensation, nuclear blebbing, and fragmentation, and
cellular shrinkage were assessed by microscopy of Cytospin (Shandon,
Sewickley, PA) preparations of 5 x 104 cells treated
with a DiffQuick stain (3). Although the data from the morphological
assessments for apoptosis are not presented here, they confirmed in all
cases the data obtained by flow cytometry.
Flow Cytometry
For quantification of both endonucleolytic DNA cleavage and loss
of DNA content, 1 x 104 cells were analyzed at 488 nm
with a FACSTAR flow cytometer (Becton Dickinson) using appropriate
filters, as described previously (77). For ddUTP labeling, control
specimens (see above) were used to determine the cut-off below which
98% of the negative cells fell.
Immunoblot Analysis
Immunoblot analysis was performed as described previously (39)
with modifications. Approximately 5 x 105 washed
cells resuspended in 2x Laemmli buffer were subjected to 10% SDS-PAGE
and transferred to nitrocellulose. The blot was blocked with a TN-TBM
solution that consisted of 10 mM Tris (pH 7.7), 150
mM NaCl, 0.01% Triton X-100, 2% BSA, fraction V (Sigma),
5% non-fat dried skim milk, and 0.01% NaN3. Antigen was
detected with a rabbit anti-mouse Bag-1 antiserum (39) at a 1:1000
(vol/vol) dilution in the TN-TBM buffer containing 1 µl normal goat
serum and 50 ng/ml ovalbumin (Sigma). Parallel control blots were
probed with preimmune rabbit serum. Antigen-antibody complexes were
then detected using enhanced chemiluminescence (ECL kit, Amersham,
Arlington Heights, IL).
Cell Transfection
For constitutive overexpression of Bag-1, full-length murine
Bag-1 cDNA was subcloned into the BamHI site of Neo-pCMV,
which contains the neomycin resistance gene and a CMV promoter/enhancer
element upstream of the BamHI insertion site. Ba/F3 cells
(1.5 x 107) were transfected with 50 µg of
linearized empty vector (Neo-pCMV) or vector containing Bag-1
(Bag1-pCMV) by electroporation (78). Stable transfectants were
generated by selection with G418, and clones were obtained by limiting
dilution. Five independent clones from each transfection were selected
on the basis of highest levels of Bag-1 expression as determined by
immunoblot analysis. Although the data shown here are from two
independent clones, comparable results were obtained for each of the
five transfectants overexpressing Bag-1.
Statistics
Quantitative analyses of Bag-1 levels were obtained by scanning
densitometry (Molecular Dynamics, Sunnyvale, CA) of anti-Bag-1
immunoblots. Unless otherwise indicated, all experiments were performed
in triplicate and reported as mean values; where not visible, error
bars (± SEM) lie within the symbols diameter.
 |
ACKNOWLEDGMENTS
|
---|
We thank Mr. Seong-Joo Jeong and the Lucille Markey Flow
Cytometry Unit at the University of Pennsylvania for their excellent
support.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Charles V. Clevenger, M.D., Ph.D., Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 509 Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104.
This work was supported by American Cancer Society Grant BE-250 (to
C.C.) and NIH Grants AI-33510 (to C.C.) and CA-67329 (to J.R.). C.
Clevenger is a recipient of an American Cancer Society Junior Faculty
Research Award (JFRA-588).
Received for publication October 10, 1996.
Revision received January 14, 1997.
Accepted for publication February 13, 1997.
 |
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