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Acta Biochim Biophys
Sin 2008, 40: 595-600
doi:10.1111/j.1745-7270.2008.00433.x
Stepping stones in the path of
glucocorticoid-driven apoptosis of lymphoid cells
E. Brad Thompson*
Departments of
Biochemistry and Molecular Biology, Internal Medicine, University of Texas
Medical Branch, Galveston, Texas 77555-1068, USA
Received: May 5,
2008
Accepted: May 9,
2008
*Corresponding
author: Tel, 1-409-772-3667; Fax, 1-409-772-6364; E-mail, [email protected]
Cumulative
work on glucocorticoid (GC) regulation of genes in lymphoid cell cultures has revealed
that apoptotic sensitivity to GCs depends on sufficient active GC receptors in
the cells. The actions of the ligand-driven GC receptor that lead to apoptosis
depend on interactions with other major cell-signaling systems, including the
MAPK pathways, the cAMP/PKA pathway, the hedgehog pathway, the mTOR system and
the c-myc system. The balance between these systems determines whether a
given cell responds to GCs by undergoing apoptosis. A central core of networked
genes may be found under GC control in many types of malignant, GC-sensitive
cells. The partial core list identified should be tested in clinical cell
samples from hematologic malignancies.
Keywords glucocorticoid; apoptosis; lymphoid cell
Early Background
How adrenal corticosteroids, specifically glucocorticoids (GCs),
kill lymphoid cells is a longstanding problem in steroid hormone action. The
mechanism of this effect is of great importance to both basic science and
clinical applications. The lympholytic effect was discovered early in steroid
research, when adrenal extracts were observed to obliterate over 90% of mouse
thymic cells. Later, it was shown that the active principles in the extracts
were GCs. The thymic cells killed by GCs were found to be immature lymphocytes
in the T-cell lineage. It is not clear whether GCs acted to kill them by a
direct effect on the lymphoid cells themselves or indirectly, by affecting
other systems upon which the viability of the immature lymphocytes depended. In
the mid-20th century, the general hypothesis of steroid hormone action, which
stated that GCs could act by regulating gene expression, developed. Evidence
obtained in vivo on the induction of certain liver enzymes, such as
tyrosine aminotransferase, supported the hypothesis [1–3], but there was still
considerable debate on the subject. The development of the hepatoma tissue
culture (HTC) cell line and other hepatoma cell lines, which responded to GCs
with increased production of the enzyme tyrosine aminotransferase [4–7], allowed more
direct tests of the hypothesis. Use of HTC cells, for example, permitted
experiments showing that GCs can act directly on their target cells to cause
transcription-dependent increased synthesis of a specific protein [4,8,9]. In
other words, no cellular systems other than the target cells themselves need be
involved for a GC to induce an enzyme. Rodent and in vitro cellular
experiments suggested that a component of control by GCs could also exist at
the post-transcriptional level [8–10]. Although later work in HTC cells
concluded that this was a secondary effect of the techniques used at the time
[11], considerable data confirm that, at least in some systems,
post-transcriptional regulation due to steroids does exist [12]. This is
especially dramatic in the control of vitallogenin synthesis by estrogens [13].
But the most basic and general regulatory effect of GCs and other steroids is
to change the level of expression of specific genes by altering their rate of
transcription. These changes in expression can be increases or decreases. Both are important for the effects of GC’s
on lymphoid cells.Given that GCs regulate lymphoid cell gene expression, the next
question to answer was “How”? The key to this question was intracellular
receptors for GCs in lymphoid cells [14]. These receptors bind steroids with
high affinity and act as ligand-driven transcription factors. Use of tissue
culture cell systems to study gene induction had already proven so valuable
that cultured cell systems for studying the lympholytic effects of GCs were
soon sought. The first reported system appears to have been S49 cells, a line
derived from a mouse lymphoma [15]. With these cells, it was shown that GCs act
directly on the target cell for liver gene induction. However, the measured
response was cell death. This allowed strong selective pressure on the cultured
cells, which in turn allowed selection of GC-resistant cells. Among these,
several classes of resistant cells were identified. Though a few resistant
clones appeared to have normal receptors (as could be best determined by the
pre-gene cloning methods then available), the majority had either lost or
mutated their GC receptor. Because the mouse cell model could only partially mimic the behavior
of human lymphoid cells, we sought a human lymphoid cell system that was
sensitive to GC-dependent cell death. In the 1970s, we screened the few
available lines of human leukemic lymphoblasts and found a useful tool in a
human lymphoblastoid cell line (CEM), which came from a case of childhood
lymphoblastic leukemia [16]. From the original line, without deliberate
selective pressure, we cloned cells that were highly sensitive to GC-driven
cell death (e.g., clone C7) and others that were highly resistant (e.g., clone
C1). The use of clonal cell stocks has allowed many studies to be done without
the confusion that often comes from using uncloned cell lines, which inevitably
include phenotypically mixed populations. Particularly interesting was the fact
that the GC-resistant C1 cells contained as many abundant high-affinity
receptors for GCs as the sensitive C7 cells. The resistant C1 cells receptors
could mediate gene induction and, therefore, were functional [17]. This
receptor-positive, GC-resistant cell phenotype was an exciting model to have
because, clinically, many human hematologic malignancies that become GC
resistant retain GC receptors [18–21].The GC-sensitive clone C7 cells also were valuable. With them, we
demonstrated again that the lethal effects of GCs are directly on the affected
cells. The extent of kill corresponded to the concentration of GCs that
occupied increasing proportions of the GC receptors, and maximal cell kill
corresponded to maximal receptor occupancy. Selection for resistant subclones
of C7 cells was done in two ways. First, in an experiment based on Luria-Delbr?k fluctuation analysis, we
used a maximal receptor-filling concentration of GCs and selected for resistant
survivors. This method, developed for analysis of prokaryotic organisms, shows
whether drug resistance results from spontaneous mutations or from non-genetic
adaptations to the drug. GC resistance in the sensitive C7 clone occurred in a
manner consistent with random, spontaneous mutations and at a surprisingly high
rate of about 10–5. This rate is more consistent
with a haploid mutation rate than with a diploid, autosomal gene and the
GC-receptor is a diploid gene. Resistance almost always appeared to be due to an
alteration in the GC receptor, giving rise to a phenotype not previously
described [22,23]. In clone after clone of these GC-resistant cells, the GC
receptors behaved similarly: they could bind steroid in vitro, but under
conditions that lead to receptor activation to its functional form as a
transcription factor, they lost the ability to retain GCs. Thus we termed such
receptors ?ctivation-labile
(actl). Later, the reason for the high mutation rate and the high frequency
of a single phenotype was found. Both CEM C7 and CEM C1 cells and, in fact, the
cells of the patient from which the CEM line came are haploid for wild-type GC
receptors [24–26]. The abnormal allele contains a point mutation in the receptor
L753F that gives the actl phenotype. The wild-type
allele mutates at the expected haploid rate, allowing selection for clones that
retain only the L753F mutant receptor, which does not mediate GC-dependent cell
death.In the second method of selection, C7 cells were first subjected to
intensive chemical mutagenesis and then selected for GC resistance. A different
phenotype predominated in the resulting GC-resistant clones [23]. The majority
appeared to lack GC-binding (receptor) activity altogether. Later it was found
that at least some of these still were expressing the actl receptor allele [27]. These combined experiments showed that, for
human lymphoid cells to die in response to GCs, GC receptors in sufficient
numbers and with normal function are absolutely required. The CEM C1 clone,
which displays GC resistance despite having sufficient GC receptors, lacks the
essential mechanisms required for the cell death response to GC. It should be
noted that, due to epigenetic instability over long-term culture periods,
expression levels of the glucocorticoid receptor in C1 cells dwindle, often
leading to populations with reduced receptors. Recloning of C1 cells allows
recovery of resistant cells with receptor quantities akin to C7 cells, which
leads to the conclusion that GC receptors are necessary but not sufficient for
GC-dependent lymphoid cell death. Subsequent studies on these cells have
provided deeper insights into the interrelated signaling pathways that support
GC action.
Apoptosis is the Type of Cell
Death Caused by GCs in Lymphoid Cells
Prior to the recognition of apoptosis as a form of cell death,
GC-dependent death of lymphoid cells was simply referred to as
“lysis” or “death”. When histological studies defined
apoptosis and the biochemical cytological correlates of cell shrinkage, DNA
lysis, membrane eversion of phosphatidyl serine etc. were uncovered, it became
apparent that GC-driven lymphoid cell death is classic apoptosis [28,29]. As
the complex caspase and Bcl2 systems, which are so
important for regulation of apoptosis, have become better understood, it is
clear that they are the ultimate regulators of this apoptosis. The caspases
have been shown to be involved in CEM cell apoptosis [30]. However, it is not
clear exactly what the step-by-step pathway is for GC-dependent lymphoid cell
apoptosis. The mitochondrial breakdown-dependent subpathway of caspase
activation is involved, but the exact steps remain to be determined.
Experiments testing for direct regulation of caspase and Bcl2 family genes suggest that GC-driven lymphoid apoptosis involves
more than direct induction of caspase or pro-apoptotic Bcl2 family genes. The induction of such genes is involved, but how
their expression and the activity of their protein products are controlled is
complex.
The Time-course of GC-driven
Apoptosis Implies a Gene Cascade
To date, results from cultured lymphoid cell lines have shown that
considerable time must elapse after the addition of GCs before the first
biochemical evidence of apoptosis can be detected. The exact interval between
initial exposure to GCs and the early stages of biochemical apoptosis varies
between cell lines, but the interval seems to exceed at least 12 h. In
GC-sensitive CEM cell clones, this lag period for biochemical apoptosis ranges
between 20 h and 48 h. Several points about the lag are noteworthy. First, GCs
must be continually present during this period for apoptosis to ensue.
Premature removal of GC or addition of a competitive antagonistic ligand to
displace the GC agonist from its receptor results in failure to cause apoptosis.
The cells then simply resume normal growth. Second, the onset of apoptosis does
not occur in a synchronized pattern. Initially, a few cells die, and gradually
more are recruited. Maximal cell death may require 12 d. Third, GCs stop the
cell cycle in a G1– or G0-like
state. This cycle inhibition may be inextricably linked to eventual apoptosis,
as in CEM cells [31], or may be dissociated from cell death, as in P1798 cells
[32]. Fourth, once the biochemical processes of apoptosis have begun, they
cannot be reversed by removing the GCs. Thus, GCs do not act as an abrupt
switch. Rather, they act as drivers of a time-dependent continuum that
ultimately triggers overt apoptosis.These facts can be evaluated in light of the known actions of GCs on
gene regulation. GCs and other steroids can regulate genes by primary, delayed
primary or secondary actions [33]. Primary gene control is relatively rapid. A
ligand-activated receptor enters the nucleus in minutes to bind at specific,
open regulatory sites on the DNA. Altered transcription and all the subsequent
steps that end in altered protein expression are completed within minutes
thereafter. From specific examples studied, new protein can be seen as early as
15 min after adding GCs to the system. As a rule of thumb, primary induction
may require up to 2 h due to requirements for RNA processing, protein folding
and the like. In some cases, primary induction may be delayed for several hours
(delayed primary induction). The mechanisms behind the delay are not well understood,
but they presumably relate to requirements for time-consuming chromatin
modifications ultimately dependent on protein complexes formed in the nucleus
between the GC receptor and a large and varied number of other proteins. No new
protein synthesis is required for either form of primary gene regulation by
GCs. In contrast, secondary regulation does require new protein synthesis. In
this type of regulation, the primary events driven by the ligand-activated GC
receptor result in production of proteins, which in turn (secondarily) regulate
genes.The behavior of cells exposed to GCs and destined for apoptosis
strongly suggests that all the above mechanisms, particularly the slower ones, are
involved. That GC exposure does not act as a brief switch tells us that the GC
receptor must continually act to modulate gene expression. It also implies the
involvement of a gene network. This network must involve interactions between
the GC signaling system and other signal transduction pathways. Interactions
between the GC and cAMP pathways have been known for many years [34]. More
recently, a wealth of data has been presented, showing important connections
between steroid-driven and other signal transduction networks [35–39]. Our work
based on CEM cell clones and extended to other cell lines, as well as that of
others, has begun to reveal this network.Before microchip-based gene array methods were available, we found
by surveying a small number of intuitively selected genes, that GCs rapidly and
selectively repress transcription of c-myc [40]. Later, we showed that
constitutive expression of c-myc partially protected against
GC-dependent apoptosis [41]. Since the protein product of c-myc is a
master regulator of transcription, our findings gave support to the hypothesis
of interactive networks in GC-driven lymphoid cell apoptosis. Adding to the
conviction that multi-gene, multi-signaling networks were working in
conjunction to drive GC-dependent apoptosis were manipulations of the cAMP
pathway. Remembering the known interdependence in lymphoid cells of GCs and
cAMP actions [42,43], we tested the effects of activating the cAMP path.
Forskolin activates adenyl cyclase, leading to elevation of intercellular cAMP
and activation of cAMP-dependent protein kinase A (PKA). We found that
activation of PKA by forskolin synergized with GCs in a clone of GC-sensitive
CEM cells, and even more impressive, PKA activation converted the C1 clone of
completely resistant cells to GC-sensitive cells [44]. We since have discovered
that a specific isoform of PKA is involved and that this cAMP/GC-pathway
interaction links to the hedgehog signal transduction pathway [45].
Studies with Large Gene Arrays
Reveal the Outlines of MAPK Pathway Interactions with GCs and their Receptors
Microchip-based gene arrays have allowed analysis of thousands of
genes for altered expression in the presence of GCs. We have employed a closely
related set of three CEM cell clones to determine which genes are best related
to GC-driven lymphoid cell apoptosis. The clones employed are: CEM-C7-14,
CEM-C1-6 and CEM-C1-15. These are subclones of the original C7 and C1 clones,
derived without selective pressure. CEM-C7-14 is inherently sensitive to GC-driven
apoptosis. CEM-C1-15 is inherently resistant and shows characteristics very
similar to those of its clonal parent CEM C1. Such recloning periodically is
necessary to preserve phenotype in long-term cultures. CEM-C1-6 is a sister
clone to C1-15 and is a spontaneous revertant to GC sensitivity. At the outset
of work with these three clones, we hypothesized that since clones C7-14 and
C1-6 are sensitive to GC-driven apoptosis, they would share a set of
GC-regulated genes distinct from those in resistant clone C1-15. This proved to
be the case. Using microchips that could detect approximately 12,600 genes and
arbitrarily setting allowable induction and repression limits high to minimize
“false positives”, we uncovered sets of 39 induced and 21 repressed
genes that were unique to the two sensitive clones [46]. The clone resistant to
GC-dependent apoptosis was not unresponsive to GCs, but for the most part
showed regulation of genes differently than those in the sensitive cells. Among
the genes uniquely repressed in the two sensitive clones was c-myc,
confirming our earlier results. The repression of c-myc is rapid and
consistent with a primary GC receptor effect. Of the genes repressed at later
times, 40% appear to be c-myc dependent (our unpublished results). Subsequent experiments revealed that an important action of GCs in
the sensitive clones is to activate p38, a member of the mitogen-activated
protein kinase (MAPK) system. We also discovered that p38 MAPK specifically
phosphorylates the serine at position 211 in the human glucocorticoid receptor
[47]. Phosphorylation of S211 enhances the ability of the
receptor to regulate transcription and apoptosis [47,48]. By using chemical
inhibitors on specific classes of MAPKs, we found that inhibition of p38
reduced GC-dependent apoptosis, whereas inhibition of the other two major MAPK
classes, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal
kinase (JNK), enhanced apoptosis. This balance was critical for determining the
sensitivity of CEM cells. The resistant clone C1-15 displayed high basal JNK
and ERK activity relative to that of p38 [49]. Inhibition of JNK and ERK
converted C1-15 cells to GC sensitive. Both forskolin and rapamycin, treatments
known to cause GC-resistant CEM cells to become GC-sensitive cells [44,50],
affected the MAPKs appropriately by reducing activated ERK and JNK and
increasing activated p38. Several pathways that modulate GC-dependent apoptotic
sensitivity converge on the MAPK pathway [51]. Many of these GC-regulated
events depend on transcription; others are post-transcriptional protein
modifications that affect enzymic activities.
An Initial Gene Profile for
Apoptotic GC Sensitivity
Focusing at first on transcription-level regulation, we examined the
responses of genes in cell lines representative of several types of hematologic
malignancies [52]. Clinically, T-lineage and B-lineage lymphoid cells tend to
differ in response to GCs and other drug treatments. Adult and childhood
leukemias also tend to differ in their responses. Malignant and normal myeloid
lineage cells tend to resist GC-driven apoptosis, but an uncommon type of
myeloid leukemia is GC sensitive. Therefore, we selected GC-sensitive cell
lines representative of several types of leukemia: pediatric T-lineage (CEM), pediatric
B-lineage (SUP-BIS), adult B-lineage (RS4; 11), and myeloid (Kasumi-1). We also
included comparisons of the effects of GCs on the genes of normal mouse
thymocytes. As a negative control, we employed the CEM-C1-15 clone. Since we
knew of several treatments that could render these cells GC sensitive, we were
able to compare the genes affected in C1-15 cells in their GC-resistant and
GC-sensitive states. The time-course of apoptotic response in the sensitive cells
conformed to the pattern discussed above. Each line displayed a lag phase after
the addition of GCs and before cell death manifested itself. For gene
expression analysis, a nested set of comparisons was performed. To avoid
missing any regulated genes of importance, no arbitrary fold-changes in
expression were imposed, and any consistent change 20% from background was
initially accepted. After categorizing the changes in expression at this level,
simple tests of statistical significance were carried out and the lists revised
accordingly. When all consistent changes in expression of mRNAs common to all
GC-sensitive CEM cells (including C1-15 cells converted to GC sensitive by
treatment with two structurally different GCs) were compiled, 96 regulated
genes were revealed. Of these, 35 were found to fit into a single network in
which c-myc and NR3C1 (the glucocorticoid receptor gene) were hubs.Additional data from the two B-lineage leukemias and the
GC-sensitive acute myeloblastic leukemia resulted in a reduced list of 27 genes
regulated (up or down) in all sensitive cell lines. The fact that these results
were derived from quite different cell types suggests a core of GC-regulated
genes common to all or many apoptotically sensitive hematologic malignant
cells. Ten of these genes were regulated in normal mouse thymocytes.
Considering the differences between the systems (growing, malignantly
transformed versus non-growing, non-transformed), it is striking that a third
of the genes were still found in common.These lists are undoubtedly incomplete. The gene microchips employed
were capable of showing only approximately 12,000 human genes and approximately
6000 mouse genes. Further studies with chips querying the entire genome will no
doubt reveal additional GC-sensitive genes common to apoptotically sensitive
hematological cells. Furthermore, other than CEM clones, only single examples
of each malignant cell type were employed. Each of these gave rise to subsets
of genes not common to all cell types. By querying other examples of each type,
it will be possible to see whether specialized gene sets can be identified that
will specifically profile each malignant cell type (e.g. adult B-cell
leukemia). An early attempt at clinical correlations with a few patient samples
has been made [53]. Even at this current level of identification, the data
encourage examination of additional clinical specimens to see whether the in
vitro results will prove applicable to prognostic evaluations in patients.
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