Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636
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ABSTRACT |
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-Adrenergic receptor (
AR) activation
and/or increases in cAMP regulate growth and proliferation of a variety
of cells and, in some cells, promote cell death. In the current studies
we addressed the mechanism of this growth reduction by examining
AR-mediated effects in the murine T-lymphoma cell line S49.
Wild-type S49 cells, derived from immature thymocytes
(CD4+/CD8+) undergo growth arrest and
subsequent death when treated with agents that increase cAMP levels
(e.g.,
AR agonists, 8-bromo-cAMP, cholera toxin, forskolin).
Morphological and biochemical criteria indicate that this cell death is
a result of apoptosis. In cyc
and kin
S49
cells, which lack Gs
and functional protein kinase A
(PKA), respectively,
AR activation of Gs
and cAMP
action via PKA are critical steps in this apoptotic pathway. S49 cells
that overexpress Bcl-2 are resistant to cAMP-induced apoptosis. We
conclude that
AR activation induces apoptosis in immature T
lymphocytes via Gs
and PKA, while overexpression of
Bcl-2 prevents cell death.
AR/cAMP/PKA-mediated apoptosis may
provide a means to control proliferation of immature T cells in vivo.
programmed cell death; protein kinase A; forskolin; Gs
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INTRODUCTION |
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EUKARYOTIC CELLS
have evolved a simple but carefully modulated pathway to regulate
formation and action of the second messenger cAMP. A large variety of
hormones and neurotransmitters bind to plasma membrane receptors and
activate heterotrimeric GTP binding (G) proteins, which, through
actions of their - and
-subunits, regulate positively (i.e.,
Gs) or negatively (i.e., Gi) the enzyme adenylyl cyclase (AC). AC catalyzes cAMP formation from ATP, and cAMP,
in turn, binds to its intracellular "receptor," the regulatory (R)
subunit of cAMP-dependent protein kinase (PKA), thereby dissociating R
subunits from the catalytic subunit of PKA. This dissociation allows
the catalyst of PKA to promote the phosphorylation of cellular proteins
involved in metabolic regulation, growth control, and differentiated
functions, such as secretion and muscle contraction. In the current
studies, we have focused on the role of cAMP in regulation of cell
growth and cell death.
Death of eukaryotic cells occurs either via necrosis or apoptosis, which are functionally, morphologically, biochemically, and mechanistically different processes. In contrast to necrosis, which is a pathological response to adverse conditions (i.e., injury), apoptosis can occur physiologically such that cells are deleted in an orderly and highly regulated manner. Apoptotic cells characteristically undergo cell shrinkage, membrane remodeling/blebbing, phosphatidylserine redistribution to the cell surface, DNA fragmentation and condensation, and formation of apoptotic bodies (3, 16, 21). By contrast, necrotic cells do not exhibit these morphological and biochemical processes. As a tightly regulated physiological process, apoptosis plays a vital role in tissue homeostasis, embryonic development, and the immune response; it can be stimulated by a wide variety of extracellular and intracellular "death stimuli," such as DNA damage, oxidative stress, hormones, cytokines, and drugs.
cAMP is a unique second messenger in that it has an antiapoptotic role
in certain cell types (4, 12, 14, 25) and is proapoptotic
in others (18, 22-23, 26-27, 30, 32). The reason
for these different responses in different cell types is unclear. In
the current studies we undertook experiments to examine cell growth and
death in response to agents that increase cAMP in the murine T-lymphoma
cell line S49. S49 cells were originally isolated from a Balb/c mouse
as transplantable T-cell tumors derived from an immature thymocyte
(CD4+/CD8+) (19, 33). Increases in
cAMP can kill S49 cells, and based on resistance to this killing,
several S49 variants with lesions in the pathway of cAMP formation and
action have been generated (33). In the current studies,
we assessed whether the death of S49 cells induced by -adrenergic
receptor (
AR) and Gs activation and/or increases in cAMP
is due to necrosis or apoptosis. By employing morphological and
biochemical criteria, we show that the death induced by
AR
activation and/or increases in cAMP is due to apoptosis and not
necrosis and that activation of Gs and AC are sufficient to
induce S49 cell apoptosis. Furthermore, our data show that PKA is also
obligatorily required for
AR- and cAMP-mediated apoptosis and that
the apoptosis induced by cAMP signaling components can be largely
blocked by overexpression of the Bcl-2 protein.
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METHODS |
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Cell culture.
Wild-type (WT), kin (which lack activation of the
catalytic subunit of PKA), cyc
(which lack expression of
Gs), neo (which were selected to be neomycin resistant),
and Bcl-2 (which overexpress Bcl-2) S49 cell lines were cultivated at
37°C in a 90% air-10% CO2 atmosphere in Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated horse
serum, 100 U/ml penicillin, and 100 U/ml streptomycin (17, 19,
33). Cells were maintained in logarithmic growth. For experiments, cells were cultured at 2 × 105 cells/ml,
and before drugs were applied, 100 µM 3-isobutyl-1-methylxanthine, a
cyclic nucleotide phosphodiesterase inhibitor, was incubated with cells
for 30 min. S49-neo and S49-Bcl-2 cell lines were created by stably
infecting S49 cells with a recombinant amphotropic retrovirus carrying
a G418 antibiotic resistance gene alone or in combination with a Bcl-2
complementary DNA, respectively (17).
Measurement of cell viability. Cell viability was determined by either trypan blue exclusion or a flow cytometric method. Trypan blue was added to cell suspensions (final concentration 0.1%), which were incubated at room temperature for 1 min, and the cells were counted with a hemocytometer. At least 100 cells were counted per sample. The percentage of viable cells was calculated by dividing the number of cells excluding trypan blue by the total number of cells and multiplying by 100. For flow cytometric analysis, cells were pelleted, washed, and resuspended in phosphate-buffered solution (PBS). Cells were examined on a Becton Dickinson FACScan using CELLQuest software (Becton Dickinson Immunocytometry System, San Jose, CA). Individual populations of cells (5,000 cells per experimental sample) were selected by gating with the use of a forward light scattering vs. side light scattering dot plot as a measure of cell viability.
ISEL+ staining. For ISEL+ staining (in situ end labeling analysis for apoptosis), we adapted a previously published procedure (6). Cells were air-dried on Superfrost Plus microscope slides (Fisher Scientific) and then incubated in labeling mix [100 mM potassium cacodylate, 2 mM CoCl2, 0.2 mM dithiothreitol, and 0.5 µM digoxigenin-11-dUTP (Boehringer Mannheim) containing 150 U/ml terminal deoxynucleotide transferase (GIBCO-BRL)]. Slides were then incubated in a humidified chamber for 1 h at 37°C. Incorporated digoxigenin-11-dUTP was detected with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Boehringer Mannheim). After slides were washed, alkaline phosphatase activity was detected by incubation in substrate solution containing 4-nitroblue tetrazolium chloride (Boehringer Mannheim) and X-Phosphate (Boehringer Mannheim). Color was allowed to develop for 1 h, after which the reaction was stopped by transferring slides into 1× Tris-buffered saline.
Agarose gel DNA electrophoresis. The cells were first washed with PBS, and their DNA was isolated using a Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). DNA concentration was determined by ultraviolet (UV) absorbance at 260 nm, and the DNA (20 µg/lane) was electrophoresed on 2% agarose gels. The gels were stained with ethidium bromide (0.5 µg/ml) for 15 min thereafter and then visualized and photographed under UV light.
Annexin V binding assay by flow cytometry. Cells were treated for 60 h, and then 0.5-1.0 × 106 cells were pelleted and washed with Hanks' balanced salt solution (HBSS) plus 1 mM Ca2+. FITC-conjugated annexin V (annexin V-FITC; 0.2 µg; Caltag Laboratories, San Francisco, CA) was added to the cells resuspended in 100 µl of HBSS and incubated at 37°C for 15 min. The cells were washed and resuspended in 1 ml of HBSS and analyzed as described for Measurement of cell viability. FITC was detected using a 530/30-nm band-pass filter (FL1 channel).
Analysis of DNA content and cell cycle by flow cytometry. Cells were pelleted, washed with PBS one time, and suspended with the addition of 2 ml of ethanol in 200 µl of PBS. Fixed cells were pelleted at 800 g and resuspended in 1 ml of PBS plus 1% bovine serum albumin. Preboiled RNase A (GIBCO) was added to a final concentration of 100 µg/ml RNase A together with propidium iodide (PI; Sigma) to 50 µg/ml. The suspension was then incubated at room temperature for 15 min and subjected to flow cytometric analysis with excitation at 488 nm and emission measured at 560-640 nm (FL2 mode).
Data analysis. Unless indicated otherwise, all experiments were conducted at least three times and yielded similar results. In general, representative results from a single experiment are presented.
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RESULTS |
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Both isoproterenol and cAMP cause cell arrest at G1 and
decrease the number of viable S49 cells.
AR activation and/or increases in the second messenger cAMP have
been shown to arrest the cell cycle progression of S49 lymphoma cells
at G1 and, ultimately, to cause the death of these cells (9, 33). As a starting point for studies with the current batches of cells, we assessed the effects of the
AR agonist
isoproterenol (Iso; 100 µM) and the cAMP analog 8-bromo-cAMP
(8-BrcAMP; 1 mM) on the cell cycle progression and cell survival of WT
S49 cells. For cell cycle analysis, asynchronized S49 cells were
stained with PI and then analyzed for DNA content by using the FACScan. We found that 42% of untreated WT S49 cells were in the S and G2/M phase of the cell cycle (Fig.
1A, top), but with
Iso treatment for 24 h, only 12% of the cells were in S and
G2/M (Fig. 1A, middle). Treatment
with 8-BrcAMP for 24 h led to 6% of the WT S49 cells in S and
G2/M (Fig. 1A, bottom). In parallel
with the cell cycle analysis, we determined the effect of Iso and
8-BrcAMP on the viability of S49 cells by the trypan blue exclusion
assay. The results (Fig. 1B) showed that WT S49 cells died
in a time-dependent fashion: cell viability declined rapidly after
36 h of treatment, with the majority of the S49 cells dead after
72 h. Together, these results confirm that
AR activation and
increases in cAMP arrest the cell cycle progression in G1
and ultimately kill S49 cells.
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Iso induces S49 cell death by apoptosis.
Because AR activation and/or increases in cAMP caused the death of
S49 cells, we conducted studies to determine whether this cell death is
due to either necrosis or apoptosis. We first examined the
morphological changes of WT S49 cells treated with Iso. Apoptotic cells
are smaller than live cells because of nuclear and cytoplasmic condensation. As shown in Fig. 1C, the trypan blue-stained
(and therefore dead) cells were smaller than the unstained, live cells (right). The observation that cell death is accompanied by
cell size shrinkage suggests that Iso-induced cell death of S49 cells is likely due to apoptosis (16, 17). We found similar
results with cAMP-treated S49 cells (data not shown). In subsequent
experiments we used 100 uM Iso because of concerns about its oxidation
during the lengthy culture period, although lower concentrations (
100 uM) were able to promote cell death.
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Gs is required for Iso-induced S49 cell apoptosis.
The observation that both
AR activation and increased intracellular
cAMP levels lead to S49 cell apoptosis suggests that the activation of
the signal cascade involving
AR, Gs protein, AC, and PKA
is proapoptotic in T lymphocytes. To define the role of these various
signaling components in S49 cell apoptosis, we used a series of mutant
S49 cells with lesions in key signaling components.
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AC activation is sufficient to induce S49 cell apoptosis.
In the above-mentioned studies, we showed that AR activation as well
as Gs
activation can induce S49 cell apoptosis. Given that Gs
activates AC, we tested whether direct
activation of AC is sufficient to cause apoptosis of S49 cells by using
the diterpene forskolin, an AC activator in these cells
(10). We found that forskolin promoted apoptosis of S49
cells, as indicated by FSC (Fig. 6,
C vs. A) and staining with annexin V-FITC (Fig. 6, D vs. B). On the basis of these
results, we have concluded that activation of AC is a sufficient
upstream signal to promote S49 cell apoptosis.
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PKA is required for AR, Gs, and AC
activation-induced S49 cell apoptosis.
Because activation of each of the signaling molecules tested thus far
(
AR, Gs, and AC) increases cAMP concentration and
promotes apoptosis, and because 8-BrcAMP was able to directly induce
S49 cell apoptosis, we hypothesized that the proapoptotic effects were
all mediated by activation of PKA. To test this hypothesis, we took
advantage of the availability of kin
S49 cells, a mutant
S49 cell lacking functional PKA activity (33). First, we
determined whether activation of
AR by Iso induces apoptosis of
kin
cells. Unlike results with WT S49 cells (Fig. 2),
Iso-treated kin
cells showed no changes in cell size
(Fig. 7, C vs. A)
and were negative for annexin V-FITC staining (Fig. 7, D vs.
B), implying that PKA is required for
AR
activation-mediated apoptosis of S49 cells. Consistent with the cell
size analysis and annexin V-FITC staining, genomic DNA fragmentation
was not observed in kin
cells treated with Iso (Fig. 5,
lane 5 vs. lane 6). In additional studies we
found that cholera toxin, forskolin, and 8-BrcAMP were all unable to
promote apoptosis in kin
cells, as assessed by cell size
analysis and annexin V-FITC staining (Fig. 7, E-J).
These results indicate that PKA is required for the apoptotic effect of
AR activation, Gs activation, AC activation, and cAMP
itself.
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Bcl-2 blocks both Iso- and cAMP-induced S49 cell death.
One of the principal mechanisms of apoptosis involves pro- and
antiapoptotic proteins of the Bcl-2 family and their ability to alter
mitochondrial efflux of cytochrome c and activation of caspases (as reviewed in Refs. 2 and 8). Because Bcl-2 is an antiapoptotic protein, we reasoned that overexpression of Bcl-2 might be protective of AR/Gs/AC/cAMP-promoted apoptosis
in these cells. S49 cells expressing the G418 resistance gene alone
(neo cells) or with recombinant human Bcl-2 proteins (Bcl-2 cells) were
untreated or treated with Iso, cholera toxin, or 8-BrcAMP. On the basis
of FSC analysis, Iso treatment dramatically decreased the cell size of
neo cells (Fig. 8, M vs.
K) but resulted in only a slight decrease in the cell size
of the Bcl-2 cells (Fig. 8, C vs. A). Similar
results were found for Bcl-2 cells treated with cholera toxin (Fig. 8,
E vs. A), forskolin (Fig. 8, G vs.
A), or 8-BrcAMP (Fig. 8, I vs. A). The
treated Bcl-2 cells showed an increase in annexin V-FITC staining (Fig.
8, D, F, H, and J, respectively, vs. B), but the increase was much less than
that in the neo cells treated with Iso (Fig. 8, N vs.
L). Unlike results with the neo cells, we also detected no
genomic DNA fragmentation of Bcl-2 overexpressing cells treated with
Iso (Fig. 5, lanes 8-11). These results indicate that
Bcl-2 overexpression in S49 cells largely blocks the apoptotic effect
of activation of
AR, Gs, and PKA.
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DISCUSSION |
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The current studies have shown that the AR agonist Iso and the
second messenger cAMP promote the apoptotic death of S49 lymphoma cells, as evidenced by DNA fragmentation and changes in plasma membrane
integrity. We have further demonstrated that this apoptosis is mediated
by a signaling pathway involving
AR, Gs, AC, and PKA.
Studies with cholera toxin (a Gs activator) and forskolin (an AC activator) demonstrated that these agents promote apoptosis of
S49 cells. We also have shown that key downstream components are
required for the apoptotic effect of particular upstream ones. Thus Iso
induces apoptosis in WT, but not in cyc
or
kin
, S49 cells, which lack Gs and PKA,
respectively; cholera toxin and forskolin induce apoptosis in WT S49
cells, but not in kin
cells. Furthermore, we have shown
that the lack of an upstream component does not impair the apoptotic
effect of a downstream one, in that cAMP is capable of inducing
apoptosis in cyc
as well as WT S49 cells.
cAMP has been shown to promote apoptosis in several other cell types,
including myeloid progenitor cells (22), leukemic cells
(27, 32), ovarian cancer cells (30),
granulosal cells (23), human B-precursor cells
(27), and rat cardiac myocytes (18). Previous
studies generally used inhibitors or related approaches to conclude
that apoptosis in response to cAMP is mediated by PKA activation.
Pharmacological inhibitors can be fraught with other problems [e.g.,
H89, a PKA inhibitor, has been recently shown to block AR
(28)], and thus definitive evidence for a role of PKA in
apoptosis has been open to question. Our use of various S49 mutant
cells lacking expression or function of distinct cAMP signaling
components, including Gs and PKA, provides "genetic" evidence that the apoptotic effects, at least in S49 cells, are mediated largely, if not entirely, via PKA activation.
In contrast to the proapoptotic effects discussed above, cAMP also has been shown to be antiapoptotic in other systems such as murine macrophage-derived RAW 264.7 cells (14), mesencephalic dopaminergic neurons (25), HL-60 cells (20), and newborn rat retina (34). Thus the effect of cAMP/PKA activation on apoptosis is tissue and cell specific. The basis for such specificity is currently unknown. As a serine/threonine kinase, PKA promotes phosphorylation of a wide array of cellular proteins in S49 cells (31) as well as other cell types. A challenge for future research is to identify the substrates whose phosphorylation by PKA promotes or inhibits apoptosis. Our results showing that overexpression of Bcl-2 prominently inhibits cAMP-induced apoptosis suggest that PKA may either directly or indirectly inhibit the function of Bcl-2, an antiapoptotic molecule, or perhaps other Bcl family members (2, 15). On the other hand, the fact that Bcl-2 overexpressing cells do not undergo apoptosis, but do show a somewhat enhanced expression of annexin V, suggests that PKA may act on other molecules in addition to Bcl-2 in initiating apoptosis. Future studies will need to be undertaken to determine interaction of PKA with Bcl-2 and other Bcl-2 family membranes in S49 cells. It is possible that PKA-mediated phosphorylation alters the function of Bcl-2 family members by affecting protein function, stability, or homo-/heterodimerization (2, 15, 27).
The current data on proapoptotic effects of agents that increase cAMP (in S49 cells) are reminiscent of previous data from examination of cell death in S49 cells by other stimuli. For example, glucocorticoids, cycloheximide, calcium ionophores, and Ca2+-ATPase inhibitors can promote apoptosis in S49 cells, and overexpression of Bcl-2 is able to partially or fully block apoptosis by those agents (5, 7, 17, 29). Such results suggest that different initiating stimuli share common components in promoting apoptosis of lymphoid cells.
The pro- and antiapoptotic effects of cAMP could have important therapeutic implications. Our data, as well as previous results, show that cAMP promotes the apoptosis of various lymphoid cells, including T-lymphoma cells, B-precursor cells, lymphoblastic leukemia cells, and myeloid progenitor cell lines (see e.g., 22, 27, 32). Such results suggest that increases in cAMP may have a role in the treatment of leukemia and lymphoma. Another setting is human immunovirus infection, in which increases in cAMP and PKA type 1 appear to contribute to immune suppression and apoptosis; this pathway has been proposed as a possible therapeutic target (1). An understanding of the detailed mechanisms by which cAMP regulates apoptosis should thus prove important in determining whether such actions of cAMP and PKA can be useful targets in the treatment of diseases in which one may wish to promote or prevent cAMP-mediated apoptosis.
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ACKNOWLEDGEMENTS |
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We thank Drs. John Reed and John Cidlowski for providing Bcl-2 overexpressing S49 and neo S49 cells, members of Jerold Chun's laboratory at University of California, San Diego, for assistance with the ISEL+ assays, Kelly Bell and Brian Torres for assistance with cell cultures, and Guoqiang Jiang and Laurie Cartlidge for assistance in preparing this manuscript.
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FOOTNOTES |
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This work was supported by grants from the National Institutes of Health, the Elsa U. Pardee Foundation, and the Falk Foundation.
Address for reprint requests and other correspondence: P. A. Insel, Dept. of Pharmacology, Univ. of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0636 (E-mail: pinsel{at}ucsd.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 April 2000; accepted in final form 28 June 2000.
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