Apoptosis
and Steroid Hormones
E. Brad Thompson
The University of Texas Medical Branch
Department
of Human Biochemistry
and Genetics
Galveston,
Texas 77555-0645
INTRODUCTION
ing the mechanisms of such a fundamental
part of
biology, with so many implications for health and disease, are driving an intensive search. Before considering the effects of specific hormones, a brief overview
of some commonly held generalities
concerning the
biochemistry of apoptosis may be useful.
Apoptosis is a term invented to describe the scattered,
apparently random deaths of cells in healthy tissues (l3). Its original and primary definition is morphological:
cells undergo shrinkage
and separation
from their
neighbors, membrane blebbing, a characteristic form of
nuclear chromatin condensation,
nuclear membrane
breakdown,
and cytolysis into condensed
apoptotic
bodies. The process evokes little gross inflammation,
though the shrivelled cells and apoptotic bodies are
phagocytosed
by surrounding
cells and macrophages.
Similar changes had been observed in rodent thymocytes after exposure to glucocorticoids
in vivo or in
vitro (4, 5) and soon after the concept of apoptosis
was introduced, lymphocytolysis
evoked by glucocorticoids was proposed as a clear-cut example. Glucocorticoid/lymphoid
systems remain particularly useful in
studying ligand-induced
cell death. Other steroid hormones also are important regulators of cell viability. In
this review I will discuss the concept of apoptosis and
its biochemical correlates in relation to the loss of cell
viability dependent on steroid hormone action.
Apoptosis is an important concept because it focuses
attention on the natural turnover of cells necessary for
proper maintenance
of a healthy organism. It distinguishes this quiet and controlled cell death from necrosis, in which certain toxic assaults on cells cause swelling, membranolysis, release of lysosomal contents, and
marked inflammation. Cell death predestined
in time
and space is also a classic event in normal ontogeny,
and this programmed
cell death in many cases also
appears to be under the control of extracellular ligands,
including steroids and molecules acting through related
mechanisms. Whether all instances of developmental
programmed cell death are apoptotic is doubtful (6-g)
and some confusion exists as to how one classifies a
given case of cell death apoptotic or not. In part, this is
because certain biochemical events, originally thought
to be invariant correlates of apoptosis, have proven
dissociable in some instances. Too often, the dangerous practice of employing only one or a few criteria,
without first establishing the bona fides of the system
being studied as authentic apoptosis, is employed.
The biochemical pathway(s) that cause apoptosis are
not known, but the obvious importance of understand0666-6609/94/0665-0673$03
00/O
Molecular Endocrinology
Copyright 0 1994 by The Endomne
DNA LYSIS
Early on, it was observed that DNA lysis occurs in
apoptotic cells, for instance in thymocytes treated with
glucocorticoids.
The resultant DNA fragments took two
forms: the greater portion in large pieces of random
size, and a smaller amount in units that were multiples
of about 180 base pairs, consistent with the cuts falling
between nucleosomes (10). The short, regularly spaced
pieces resulted in “ladders” when the DNA from apoptotic cells was electrophoresed
on gels. Such DNA
laddering has been observed in many instances of
apoptosis, and it has been theorized that it is a critical
step in the process, setting off a search for “the”
endonuclease
involved (10-14). While it may well be
true that for certain examples of apoptosis the action
of a unique, specific internucleosomally
cutting endonuclease is essential, such a requirement
may not be
universal. In various examples,
DNA laddering and
apoptosis have been found dissociated. Thus, examples of apoptosis have been described without formation of DNA ladders (15-I 9). Laddered DNA has been
found in condensed chromatin, but this too may vary.
At the ultramicroscopic
level, the condensed chromatin
in apoptotic 3T3/1 OT1/2 cells differs from that of apoptotic thymocytes, perhaps reflecting differences in DNA
biochemistry (17). Indeed, perinuclear chromatin condensation may not be universally associated with nuclease activity (19). There are at least two reports of
necrosis accompanied
by formation of DNA ladders
(20, 21). Application of zinc to rat thymocytes prevents
dexamethasone-induced
DNA ladder formation
but
causes loss of cell viability (22). Not even in all cases
of glucocorticoid-evoked
lysis of lymphoid cells is DNA
laddering a prominent or early event (Refs. 19 and 23
and E. B. Thompson, unpublished results). It does seem
that some form of DNA lysis occurs universally during
apoptosis, and that, often, it takes the form of internucleosomal cuts. In some cells and systems, however,
the predominant
effect results in DNA fragments of
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large, nonsystematic sizes. In sum, DNA lysis in apoptosis need not be internucleosomal
and may be a
terminal, rather than an initiatory, event. It is certainly
unwise to classify-as
some have-a
given system as
apoptotic or not on the basis of observed DNA laddering
alone (17, 18).
CALCIUM
INFLUXES
Glucocorticoids
cause a sustained influx of Ca++ ions
in young rodent thymocytes, which contain a Ca++dependent endonuclease. These facts, and similar findings in other cells, led to the proposal that thymocyte
apoptosis is the result of Ca++ influx leading to the
activation and/or induction of a Ca”-dependent
endonuclease (24). While this sequence may explain specific
cases of apoptosis, its utility as the universal explanation of the process is in doubt, since reports have
appeared showing dissociations
between apoptosis
and Ca++ influxes (25-30).
Paradoxically,
sustained
Ca++ influxes may be seen after stimuli to cell growth.
And in T cell hybridomas or thymocytes, either glucocorticoids or activation of the TCR/CD3 complex cause
both apoptotic death and Ca++ influxes, but when both
stimuli are used, they still cause Ca++ uptake while
antagonizing each other with respect to apoptosis (31).
Shifts in intracellular
pools of Ca++ rather than net
influxes may be important for the glucocorticoid
effects
in some lymphoid cells (32). In HL60 cells undergoing
apoptosis, no early influx in Ca++ was observed (33,
nuclease has been
34). At least one Ca “-independent
shown to cause DNA ladders during apoptosis (13). No
doubt Ca” is an important regulatory molecule, but it
should be realized that, as a recent review attempting
a synthesis of its actions in cell death states, “. .cell
death can occur without any apparent change in [Ca”]
and that mechanisms other than Ca++ overload are also
important. . .” (26).
MACROMOLECULAR
SYNTHESIS
One early tenet of the biochemistry of apoptosis was
that macromolecular
synthesis was required. This view
may have stemmed from the basic knowledge
that
glucocorticoids
both cause thymocyte apoptosis and
are well known gene inducers. The evidence for required macromolecular
synthesis mostly comes from
use of inhibitors, e.g. actinomycin to block RNA synthesis and cycloheximide to block protein synthesis. Since
such agents themselves must ultimately prove lethal to
cells, a biochemical marker that correlates with apoptosis is often used as the endpoint, although sometimes an indirect measure of cell viability such as dye
exclusion is employed. Clearly great care must be used
in interpreting the results of this type of experiment. At
the least, one should establish whether there is an
absolute linkage between the chosen marker(s) and
apoptosis in the system studied. Usually, inhibitor action
is not without side effects, and these may interfere with
the interpretation of the results. For example, cycloheximide may block RNA as well as protein synthesis. Also,
in some cells a significant part of protein degradation is
protein-synthesis
dependent (35); yet the contribution
of such effects is seldom considered in experiments
concerning the inhibition of apoptosis by inhibiting protein synthesis. Furthermore, it has been reported that
treatment of certain cells with inhibitors of macromolecular synthesis causes apoptosis (36). Finally, some
systems display apoptosis without a requirement for
macromolecular
synthesis. A notable example is the
ceil death caused by treatment of cells with antibodies
to the membrane antigen Apol/Fas (35,38).
OVEREXPRESSION
OF GROWTH
GENES
Overexpression
of several genes known to be important for cell growth and the maintenance of the cell
cycle has been shown to lead to cell death. If a substance required for growth, such as interleukin-3
or
serum, is withdrawn, overexpression
of c-myc becomes
lethal to the relevant cells (39,40). It has been proposed
that c-myc simultaneously
stimulates specific intracellular signals for growth and death, and that the latter
must be blocked by expression of certain genes under
the control of the appropriate growth factors in order
for the cells to avoid death. On the other hand, expression of c-myc causes resistance to the antigrowth
effects of interferon in fibroblasts (41) and to glucocorticoids in lymphoid cells (see below). Expression of cjun and c-fos in lymphoid cells deprived of critical growth
factors also has been proposed as a requisite for apoptosis (42). Continuous expression of c-fos appears to
precede terminal differentiation
and cell death in viva
(43). Expression of the tumor-suppressor
gene p53 has
been associated with the propensity of cells to undergo
apoptosis (44, 45). One of the more constant findings
associated with apoptosis is the ability of high expression of bc12 to inhibit the process. Although not universally, apoptosis evoked by a wide array of agents,
including steroids, is blocked in cells expressing high
13~12 levels (45-51). How this protein, known to be
associated
with mitochondrial,
nuclear, and inner
plasma membranes, protects against such a variety of
insults is of great interest. One recent suggestion is
that it shields against oxidative damage (52).
Expression
of Other Genes
The basic apoptosis theory has been that the lethal
instigator activates or induces genes that are destructive to the cell. In addition to the specific examples
noted above, increased expression of the genes for
transforming growth factor-j31 (TGFPl), clusterin, various proteases, tissue transglutaminase,
and various
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667
others have been described in steroid-dependent
apoptosis. Some of these, as TGFPl, are known to be
inhibitory or lethal to certain cells under the proper
conditions. Additionally,
differential screens of cDNA
libraries have produced candidates
for apoptosis-related genes (53, 54).
GLUCOCORTICOIDS
AND APOPTOSIS
One of the earliest observed effects of glucocorticoid
treatment was thymic involution in rodents; that this
was due to an action directly on the thymic lymphocytes
was soon shown in vitro. Somatic cell and molecular
genetic studies in lymphoid cell lines have established
beyond reasonable doubt that intracellular glucocorticoid receptors are the mediators of the lethal effect
(55-59). In a sensitive cell system, glucocorticoid receptor (GR) concentration
affects the level of sensitivity
(60). Lymphoid cells show that while necessary, the GR
alone is not sufficient to convey the signal for apoptosis
(61-63) and some lysis-resistant cell lines have very
high GR content. For a time, it was thought that only
immature thymic lymphocytes underwent apoptosis in
response to glucocorticoids,
but now it is clear that
some B-derived cells also are susceptible, e.g. myeloma
and chronic lymphocytic leukemia cells (50, 64, 65).
This implies that various normal lymphoid cells are
sensitive to steroid-evoked
apoptosis, depending
on
their ontologically determined expression of the mysterious “lysis functions.” The reasons why various GR+
lymphoid cells differ in sensitivity to glucocorticoids
should yield important information into the pathways of
apoptosis. One suggestion that has been offered is that
the level of expression of bc12 determines sensitivity
(66). Further examples should be examined to test this
hypothesis.
The role of the GR in mediating lymphoid apoptosis
has been examined in cells selected for glucocorticoid
resistance and by transfection studies designed to map
the regions of the GR required. In both mouse and
human lymphoid cell lines shown to acquire dexamethasone resistance at a haploid rate, it was found that
there had been prior loss of one autosomal GR allele in
the parental clone; then a mutation in the remaining,
wild type allele led to resistance (55, 57, 58, 67, 68).
Transfection of expression vectors carrying GR genes
has been used to map the GR for its cell kill functions.
Transient transfection
of holo-GR into GR-deficient,
glucocorticoid-resistant
mutants of the human lymphoid
cell line CEM showed that replacement
of the GR in
physiological
amounts restored
steroid-evoked
cell
death (69). Deletion of the steroid binding domain
yielded GRs that were constitutively lethal and as potent as holo-GR plus steroid. The amino-terminal,
transactivation domain did not seem critically important, but
at least part of the glucocorticoid
response elementspecific DNA binding domain was required (70). Among
the more interesting GR mutants was one in which a
frame-shift interrupted
the second zinc finger of the
DNA binding domain, replacing the rest with unrelated
amino acids to yield a truncated protein. This mutant
GR was both constitutively active and fully potent (70).
In the S49 mouse lymphoid cell system, stable transfectants of GR-deficient cells indicated that the aminoterminal transactivation
domain was important for evocation of cell death (67). Surprisingly, in that system,
transfection
into cells containing wild type GR of an
amino-terminal
truncated
GR from steroid-resistant
cells enhanced rather than blocked glucocorticoid sensitivity. The differing results as to the need for the amino
transactivation
domain may be species, cell type, or
system-dependent
or may be more quantitative than
qualitative, since in the CEM cell experiments, constitutively active GR mutants containing the domain were
slightly more potent than those lacking it (70). The depth
of information available on the actions of the GR makes
pursuit of such systems one of the more promising in
understanding
mechanisms of apoptosis.
In S49, P1798,697, and CEM cells, one of the earliest
and most dramatic effects of glucocorticoid
treatment
is the down-regulation
of c-myc mRNA and protein (50,
54, 71-73)
which mechanistically may occur at transcriptional or posttranscriptional
levels (74, 75). When
GR-deficient Jurkat cells were transfected with either
holo-GR or LS7 (a mutant GR that could not transactivate from an MMTV promoter) c-myc repression and
apoptosis occurred (Helmberg and M. Karin, personal
communication).
The myc decrease is followed some
hours later by overt apoptosis. This decrease had functional significance, since transfecting susceptible CEM
cells with c-myc expression
vectors inhibited dexamethasone-induced
apoptosis. Also, down-regulation
of c-myc by antisense oligonucleotides,
in the absence
of glucocorticoids,
produced equivalent cell death (76).
These results therefore differentiate
these systems
from those in which overexpression
of myc causes
apoptosis. In S49, but not CEM cells, glucocorticoid
treatment also reduced the levels of other oncogenes,
c-myb and c-Ki-ras (72).
Some protection against lymphocytolysis
by glucocorticoids is afforded by serum (77, 78). The reasons
for this effect are not known, although GR levels were
lower in CEM cells grown in serum than those grown
serum-free (78). Bc12 also has been shown to protect
against the apoptosis evoked in lymphoid cells by glucocorticoids (50) where, as noted above, down-regulation of c-myc levels seems an important part of the
process. In other systems, the gene also has been
shown to protect against the death caused by overexpression of c-myc (49). An understanding
in biochemical
terms of this paradox would greatly aid our understanding of apoptosis in general.
Tissue transglutaminase,
which cross-links various
proteins, is induced in glucocorticoid-treated
thymocytes (79) and it has been suggested that the resultant
cross-linking of cell proteins accounts for the formation
of apoptotic bodies and failure to release chemotactic
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peptides that would otherwise
attract inflammatory
ceils
(80).
The effects of glucocorticoids
on neural tissue are
mixed. In the adult brain, the viability of many cells
seems unaffected. On the other hand, glucocorticoid
overload has been observed to cause death of rat
hippocampal neurons, and survival of cultured hippocampal cells is shortened by corticosterone
(81). It has
been suggested that under physiological
conditions,
the levels of glucocorticoids
routinely encountered may
“endanger” the nerve cells, so that exposure to additional metabolic stresses leads to cell death. The cell
death of hippocampal
neurons brought about by glucocorticoids has been dismissed as being apoptotic,
due to lack of certain biochemical criteria (82). In the
light of the uncertainties regarding these criteria, perhaps the issue should remain open.
That glucocorticoids
may synergize with other metabolic or signaling pathways to cause apoptosis
is
suggested by recent studies that link the action of
steroid hormones with activation of the protein kinase
A and protein kinase C pathways (83) and by a synergism for cell kill between CAMP and glucocorticoids
in
lymphoid cells (84, 85).
SEX
STEROIDS
removal (95, 96). TRPM-2 also rises in response to
pressure atrophy of the kidney and may therefore be a
general marker for certain forms of tissue damage (94).
It is not inextricably linked to tissue damage, since it is
expressed constitutively
in the epididymis and other
tissues and is not well correlated with the death of
neurons during development
(79). Its exact functions
are not fully known. When glucocorticoids
are used to
block apoptosis in the prostate, the TRPM-2 rise is
blocked, as are the increases in hsp 70 and c-fos seen
after castration (93). TGF@ increases in the first day
after androgen removal (96), and it has been suggested
that its ind%:on
conveys a paracrine signal for apoptosis. Direct addition of TGFP can mimic the effects of
androgen removal on the prostate (97). However, TGFP
did not prevent the prostatic regrowth stimulated by
androgen (97), suggesting counterbalancing
activities.
Bc12 levels may play a role in preventing prostatic
apoptosis. As in other systems, it has been noted that
bc12 levels increase as prostatic cancers become androgenindependent
(98). An effort to identify “castration-induced”
proteins or genes associated with apoptosis in the prostate has been made by use of several
methods, including subtraction gene libraries, A score
of proteins and several genes have been identified
which may prove relevant (54, 79).
AND APOPTOSIS
As classic an observation in endocrinology
as glucocorticoid-evoked
lymphoid ceil death is the involution of
many tissues dependent on sex steroids when those
hormones are removed (86, 87). The stimulus to research of the apoptosis concept has led to identification
of many markers for apoptosis in such tissues.
PROSTATE
In the prostate, the glandular epithelium
shows the
morphological changes of apoptosis beginning about 3
days after castration (88). In days 1 and 2 post castration, there is internucleosomal
DNA fragmentation and
Ca++ influx. This timing has led to the proposal that in
this system, these biochemical events are causal for
the apoptotic process (89). Antiandrogens
or androgen
withdrawal induce apoptosis in the prostate, in prostate
organ cultures (90) androgen-dependent
prostatic carcinomas, and in rat and human prostate-derived
cell
lines in vitro or borne as xenografts (reviewed in Refs.
79 and 91). Replacement of androgens can restore the
prostatic epithelium, although testosterone
does not
seem as fully effective as dihydrotestosterone
(92).
Glucocorticoids
repress apoptosis in the prostate (93).
Several specific genes/gene products have been associated with the apoptosis that follows androgen removal. One prominent marker is TRPM-2, also known
as sulfated glycoprotein-2
or clusterin (79, 91, 94, 95).
TRPM-2 levels increase within a day after androgen
UTERUS/OVIDUCT
The steroid-dependent
cyclical regression of the uterine
epithelium is another example of apoptosis occurring
subsequent to hormone removal (99). Estrogen stimulates uterine epithelial growth; progesterone
stimulates
its differentiation.
Removal of progesterone
or use of
antiprogestins
results in morphological apoptosis of the
epithelial cells without gross changes in the stroma
(100). Interestingly, prolonged antiprogestin
exposure
in rats resulted in continued apoptosis of epithelial cells
accompanied
by invasion by granulocytes: a hallmark
of inflammation not usually associated with the apoptotic process (101). In the monkey, RU486 caused
apoptosis associated with increased diapedesis of leukocytes (102). That familiar biochemical correlate of the
process, DNA lysis into ladders, is seen. It has been
reported that adding TGFPl to primary cultures of
rabbit uterine epithelial cells causes apoptosis (103)
although at the time of writing this review, no report of
increased TGFP in uterus after steroid manipulation
was found. In the perimplantation
mouse uterus, TGFPl
and p2 were expressed in epithelial cells, and estradiol- but not progesteroneinduced TGFP2. No mention
of apoptosis was made in these studies (104). Treatment with the potent progestin, medroxyprogesterone
acetate, caused slight, variable, and slow decreases of
TGF@ (105). Thus, the role of TGF/3 in uterine apoptosis
remains to be established. Interestingly, in VOX mice,
treatment with estriol resulted in brief stimulation of
epithelial cell growth, followed by extensive death that
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669
morphologically
was a mix of apoptotic and necrotic
features (106). Recent progress in developing tissue
culture lines of uterine cells may be helpful in studying
the biochemistry, cell biology, and molecular biology of
the process in uterine cells. In the baboon oviduct,
estradiol withdrawal
or progesterone
administration
caused apoptotic epithelial regression (107).
the normal gland, and considering the known interactions between
epithelium,
extracellular
matrix, and
stromal cells, it seems that a full understanding
of in
viva apoptosis will have to take into account genes and
gene product interactions from several cell sources,
responding to a complex network of hormonal forces.
SUMMARY
MAMMARY
AND HYPOTHESIS
GLAND
It has been known for many years that mammary gland
requires the interplay of several hormones for its proper
maintenance (108). Also crucial are interactions with
nearby stromal cells and extracellular matrix (109). Thus
for both normal mammary epithelium and hormonedependent mammary carcinomas, multifactorial
regulation must be considered (110). Estrogens, progestins,
and glucocorticoids,
the steroid hormones that are
known to affect mammary growth and development,
seem to exert both direct effects on mammary epithelial
cells and indirect effects, either by altering growth/
vitality factors produced by the epithelial cells themselves or by surrounding
cells of mesothelial origin.
Such factors may include elements of the matrix itself.
The effects of steroid hormones on apoptosis in this
tissue have been reviewed recently (79). As in the
prostate, members of the TGFfl family have been implicated by various correlations
between their levels
and hormonal manipulations
as well as by direct application (79, 109). The precise mechanistic significance
for mammary epithelial cell apoptosis of the changes in
TGFP has not as yet been established.
Evidence for
endonucleolytic
activity and altered calcium homeostas/s in mammary epithelium is not extensive. However,
treatment of human mammary adenocarcinoma
cells
(BT-20) with tumor necrosis factor causes Ca++ influx
and DNA fragmentation (111). Since in some mammary
cancers tumor necrosis factor can be regulated by
steroids, this finding may be of clinical relevance.
Screening of differential gene libraries from lactating
and involuting mammary glands has led to the isolation
of several well known markers for apoptosis: SGP-2,
tissue transglutaminase,
~53, and c-myc, as well as a
number of novel genes, still in the early stages of study.
In the involuting mammary gland (and the prostate),
destruction of the extracellular matrix surrounding
the
dying epithelial cells is seen. This is associated with the
production of a number of extra- and intracellular proteases (79). In addition, the fibroblasts of the involuting
gland show high expression of stromolysin-3, a matrix
proteinase,
implicating
extracellular
matrix modeling
(112).
Mammary cancer cells in culture demonstrate that a
variety of factors that regulate cell growth and vitality
may be affected by hormonal manipulation (109). These
may vary with the capacity of specifically differentiated
cell lines to respond to the hormones and produce/
respond to the factor. Extrapolating
this information to
In ways particular to specific cells and tissues, steroid
hormones cause apoptosis by their presence or their
absence. These apoptotic events can be initiated by
the direct action of the steroid on the affected cell or
indirectly, by altering expression of paracrine effecters
in the affected or in supporting stromal cells.
As yet, no single causative biochemical event is
adequate as a universal mechanistic explanation
for
apoptosis.
Probably, multiple biochemical
pathways
can result in morphological
apoptosis. Whether various
initiating pathways converge on a final, late, and lethal
path remains to be determined.
In the specific cases of apoptosis evoked by the
addition or removal of steroid hormones, there is a
universal requirement for the appropriate
intracellular
hormone receptors. Induction and/or down-regulation
of certain genes has been observed with some frequency in several systems. Exactly how these, or other
newly discovered genes lead to or block apoptosis
remains to be seen.
Although some form of DNA lysis always seems to
appear (at least as a late event) in cells dying apoptotic
deaths, internucleosomal
DNA cleavage is not universally observed. It has been reported, however, in steroid-related apoptosis of thymus, prostate, breast, and
uterus. Whether it plays a causative role is still open to
question. The same may be said for gross Ca++ influxes
into the apoptotic cells.
In some systems, reduction in expression
of the
protooncogene
c-myc also leads to apoptosis. Since
overexpression
of a number of growth genes, including
c-myc, under the special circumstance of simultaneous
withdrawal
of essential growth factors can lead to
apoptosis, it has been proposed that the growth gene,
e.g. c-myc, simultaneously stimulates genes for growth
and for death. It has been proposed that the death
gene induction must be countered by signals turned on
by the withdrawn
growth factor. A difficulty with this
hypothesis is that overexpression
of several growth
genes appears to cause apoptosis. It is hard to imagine
how each of them can turn on the same lethal pathway.
Do jun, fos, ~53, myc, (and no doubt other yet to be
elucidated cell growth genes) all have direct control
over a singular apoptotic path? Or does each of them
have control over an independent lethal program? Similarly, are there one or many countervailing
programs
under the control of various growth factors?
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670
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END0.1994
This work was supported
in part by National Cancer Instrtute (Grant 5 ROl CA41407)
and National Institutes of Health
(Grant NIDDK-5
PO1 DK42788).
HYPOTHESIS
I propose an alternative mechanism: that cells are able
to recognize an imbalance between signals to grow and
not to grow. It may be the imbalance that is recognized,
rather than offsetting specific pathways of lethality/
vitality genes under the site-specific control of various
growth factors. Recognition
of the discordance
between signals to grow and to stop is likely to involve
the balance that must be maintained in the complex
sequential network of cell cycle regulatory protein(s).
Their activities are often controlled by posttranslational
modifications (e.g. phosphorylation)
and protein-protein
interactions. This network is shared and made use of
by all the hormonal, growth gene, cell cycle, and growth
factor systems known. It can be influenced by transcriptional and posttranscriptional
events. This general
model offers an explanation of how c-myc up-regulation
and down-regulation
both can cause apoptosis. In the
up-regulation
experiments apoptosis obviously occurs
during withdrawal
of a growth stimulus, In the downregulation experiments, the same “push-pull” of mixed
signals may occur; only in those cases the required
growth stimulator withdrawn is Myc, and the “push” of
growth stimulation may be provided by the constitutive
expression of other growth genes. In the CEM C7 cell
system, candidates are Ki-ras and raf, both of which
are expressed constititively in the presence of dexamethasone, while c-myc levels plunge in response to
the glucocorticoid
(54, 74). The suggestion that the
network of regulatory phosphoproteins
goes awry in
consequence of mixed grow and no-grow signals can
be tested. Its consideration
adds an additional dimension to the present focus on finding new genes increased during apoptosis.
Note Added in Proof
Several papers relevant to this hypothesis have been publrshed
very recently. A short report from Ohoka et a/.(1 13) states that
the phosphatase
inhibitor okadaic acid blocks glucocorticoidal
apoptosis
in T cell hybridomas.
Several new findings
about
6~1-2 and related proteins point to the importance
of balance
between protein partners in its control in apoptosis.
Bcl-2 has
been found to be a homolog
of ted-9,
a cell survival gene in
the Caenorhabditis
elegans programmed
cell death pathway
(114) and another
gene related to bcl-2, named bcl-x has
been found in chicken and human (115). 6~1-2 and the long
form of bcl-x promote
cell survival.
The short form of bcl-x
and Bax, a short homolog
of EC/-~ promote
cell death (115,
116). Bax and SC/-2 form heterodimers
(116). Thus these may
be examples
of the signal balancing
by protein-protein
interactions I have proposed.
Acknowledgments
Received
Received
March 1, 1994. Revision
21,1994.
Accepted March 21, 1994.
Address requests for reprints
to: E. Brad
received
March
Thompson,
De-
partment of Human Biochemistry and Genetics, University of
Texas Medical
veston, Texas
No. 6
Branch, 601 Basic
77555-0645.
Science
Building
F45, Gal-
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