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Programmed cell death
Pergemon Press
Life Sciences Vol. 15, pp . 1549-1565 Printed in the U.S .A .
MINIREVIEW PROCRAMIO;D CELL DEATH Richard A. Lockshin Department of Physiology, University Dentistry, Rochester, N.Y . 14642 and de Clermont, Centre Scientifique des
1.
Definitions :
and Jacques Beaulaton of Rochester School of Medicine and Dipartement do Zoologie, Universiti Ciseaux, 63170 Aubiàre, France .
The term Programmed Call Death refers to several phe-
nomena in development, ageing, and pathology:
a finite limitation on the
number of generations cultured mammalian cells undergo (61), loss of cells during ageing, either sporadically (11,20,110,121,139) or in major episodes (12-19,27) ; or the destruction of large numbers of calls at specific points
of development (86,155) such as the loss of neurons in ganglia as others form synaptic connections (1,88) .
Other uses of the term include the normal,
exaggerated, or unusual loss of already-formed cells in developmental mutants such as rumplessness and apterous in Drosoahila (21,22,124,159,160) ; lytic responses of cells to essentially non-pathologic or developmental stimuli (70,71) including the appearance or disappearance of hormones (41-45 ;60,66,68, 69,115,136,148,156) ; and processes of dying known to include a complex sequence of identified, ultimately irreversible steps (93,125,126) .
The
latter categories include a vast number of examples, the range of which has been admirably summarised by GlUcksmann (49) and most recently revie"d by Saunders (125) .
Although the term "programmed" should most logically be
reserved for those instances in which a sequence of steps has been demonstrated, adequate documentation exists only for a few instances, and most workers have extended the concept to include what more correctly would be described as Itphysiological cell death" .
By this term we refer to the collapse of specific
cells and tissues in the absence of obviously toxic stimuli .
Acutely-killed
cells undergo a markedly different type of death, referred to as coagulative necrosis (78,79), which includes osmotic collapse or other processes of immediate disorganisation.
The definition suggested could include keratinisa-
tion of epithelial cells and cell death during normal turnover processes-essentially a cellular equivalent of metabolic turnover .
Although these
phenomena are relevant and interesting in their own rights, they are sufficiently different from the common concept or inaccessible to study to 1549
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Programmed Cell Death
warrant exclusion from a short review .
We will therefore concentrate on the
massive and controllable instances of cell death during development--situations which are most amenable to study and which provide a vantage point from which to relate the events of programmed cell death to the more general question of call loss from genetic, pathological, and temporal causes . 2.
Signalling mechanisms :
The great bulk of research in this area
has been concerned with the identification of instances of cell death and recognition of the immediately antecedent extracellular or physiological triggers .
The triggers, or controls, can be roughly divided into two
categories on the basis of the location of the control mechanism. logical regions of cell death,
Embryo-
such as in sympathetic ganglia (1) and avian
and mammalian limbs (70,71,76,125) appear to be controlled by locally diffusing materials .
The chemical nature of two substances which PREVENT cell
death (nerve growth factor and the substance which protects the cells of the posterior necrotic zone in chicks) has been established (1,26) and similar factors have been hypothesized but not identified in such situations as the bidirectional communication between neurons and their target organs .
Whether
or not these controls are routinely supportive, the absence of the control leading to involution, has not been determined .
More massive instances of
cell death are frequently mediated by more conventional hormones .
Such situa-
tions range from the involution of the Mullerian ducts under the influence of testosterone and duct-organizing substance (17,74,127-9) to the cataclysmic events of amphibian or insect metamorphosis (68,149-151 ; Table 1) . Although the supportive role of nerve growth factor may ultimately prove to relate to nutrition, inasmuch as the compound bears structural and functional similarity to insulin (1) other endocrine or diffusing chemical sig nalling mechanisms are not clearly related to intermediary metabolism, except perhaps for the lympholytic effect of cortisol (109) .
Other known signals are
idiosyncratic in nature and are not predictable in phylogenetic or other terms . Thus, in amphibia, thyroxin promotes the destruction of the tail musculature while stimulating the growth of the legs (151) ; in different species of beetles, the gonadotrophic juvenile hormone may provoke the development or the destruction of flight muscles, depending on the biology of the insect in relation to its repr9duction (16,33,136) . sexual tissue .
Similar arguments obtain for secondary
It is evident that the extracellular signals represent
an exploitation of accessible and controllable information, and that there is probably no clear order or reason to the type of signal utilized (93,
123) .
Any generality which could be drawn would relate, as for any developmental event, to the mechanisms of timing, recognition and response, and evolutionary adaptation of the physiological control mechanism .
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Intracellular avenu e
Programmed Call Death
155 1
Despite the many efforts to recognise the
axtracallular or physiological controls, surprisingly little attention has been devoted to the intracellular response phase of cell death (15) .
By far
the major number of studies are either documentation that cell death has occurred or morphological observations of the dying calls (Table 2) .
Focus
on the intracellular changes has for evident reason been limited primarily to situations of massive involution, such as amphibian and insect metamorphosis (36, 90, 91, 93, 151) and the collapse of mem ary and uterine tissue upon withdrawal of the supporting steroids (38, 156) .
Nevertheless, from thane
studies, several generalisations my be drawn, a)
Cell death normally affects specific cells and tissues, leaving
intact other, sometimes similar calls in the immediate vicinity .
Thus it
is unlikely that an extrinsic toxic substance provokes the collapse of the calls . b)
The dying cells themselves undergo considerable evolution prior to
a stage in which they lose control over their intracellular environment and amll or disintegrate (15, 142-145) .
Trump has underscored the major differ
ences between cells dying of insult such as anoxia and cells-falling prey to more biological phenomena (142-145) .
Karma, in emphasising the univer-
sality of a phenomenon he terms "apoptosis" (essentially a form of fragmentation of a dying cell) in a wide range of developing, involuting, malignant, and non-lethal pathologic situations, comments that apoptosis may be "possibly the only mode of controlled call death" (82) .
Although his generalisation
appears to be a bit too broad, the distinction is probably valid and forms the basis for the second generalisations
that there is no evidence that cells
dying in non-pathologic situations are suddenly cut off from vital supplies . Their evolution rather suggests that the intracellular milieu, as well as the extracellular, is under control and that the calls, although beating a retreat, are doing so in an organised and physiological manner .
There is
in fact some direct documentation for this statement, as is discussed below in section 4 and (103) . c)
Many forms of control, where studied, involve the synthesis of
RNA and protein prior to the beginning of involution .
Thus, antimetabolites
interfere with the collapse o£ tadpole tail tissues (140, 151), insect muscles (92), and thymocytes treated with glucocorticoids (108) .
Blockage
of protein synthesis does not appear to interfere with the activation of lytic (lysosomal) enzymes (25, 65) although the appearance of primary lysosomes may be blocked, as is discussed in section 4 below .
In the case
of thymocytes exposed to cortisol, the newly-synthesized protein say interfere with glucose uptake (108) ; elsewhere, the function of protein synths-
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Programmed Cell Death
Vol . 15, No . 9
sis is not known. d)
In a fear instances (12-14, 34, 66, 136,
nuclear lesions are reported .
147, 152) various primary
In this form of cell death, which is not
the most common, cell collapse without marked lysosomal involvement is normally followed by phagocytosis .
In other systems, the nucleus appears
to remain at least morphologically normal well into the lytic phase (Table 2) . e)
A fifth generalization is that the primary intracellular changes
which switch the cell to a catabolic state are in no sense known= nor is it certain what enzymes mediate the catabolism .
This statement is inten-
tionally and perhaps overly provocative, but is intended to counter an impression which pervades the literature on this subject .
In most but not
all tissues studied in any detail, the presence of an evolving lysosomal system
has been sought and found, and the lysosomes therefore invoked to
explain the tissue degradation .
Nevertheless, in many tissues primary changes
occur in the cytoplasm and these are on occasion flagrantly lytic (Table 2) . None of these changes have as yet been adequately explained . The significance, import, and limitations in these generalizations can be most effectively considered by examining a specific instance of cell death. Such an example could be the intersegmental muscles of silkmoths, for which a relatively broad range of information is now available (Table 3) . 4.
Illustrations
the intersegmental muscless
The intersegmental muscles
are larval muscles which are retained through the pupal stage until the emergence (ecdysis) of the adult insect, whereupon they begin to degenerate . These muscles are almost totally resorbed within 48 hours.
The signalling
mechanism involves an endocrine step at the outset of development which potenThree weeks later, ecdysis is
tiates or sets the stage for a future response .
initiated by a neuroendocrine signal (141) which activates a specific behavioral pattern including first hyperactivity and then quiescence of the intersegmental muscles (94) .
Same aspect of this change in motor activity appears to be the
proximate signal to initiate lysis (Table 3) .
Thereupon follows a period
involving the synthesis of new RNA and protein (92, 98) and the appearance in the tissue of large numbers of lysosomes (97) . are detectable biochemically slightly earlier (99) .
The lysosomal enzymes Large-scale breakdown
of the myofilamenta is recognized at an ultrastructural level within seven hours .
Lysosomes seem to be preoccupied with the destruction of mitochondria
and do not include myofilaments in their matrices .
During this phase input
resistance gradually increases and acme fragility (marked by a drop in capacitance when the fiber is placed in Ringo* appears (95, 96) but otherwise active and passive properties of the call membrane are normal . ating fiber is contractile .
The degener-
Approximately 12 hours after the emergence of
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Programmed Cell Death
the animal, autophagic vacuoles are seen in the tissue, though again they do not include myofilaments, and a phase of apoptosis or fragmentation is initiated.
It is only after this latter phase that the membrane potential
collapses.
Muscle proteins can be detected in the blood as early as three
hours after ecdysis, though they reach a peak during the height of fragmentation and collapse of the membrane potential,
24 hours after ecdysis (95) .
By 40
hours, only pycnotic nuclei and wisps of cytoplasm are left in the cell . Thus the breakdown of the intersegmental muscles involves the reception of two signals, synthesis of an unidentified protein, and activation of lysosomal and non-lysosomal lytic mechanisms .
Morphologic signs of nuclear
failure occur only at a relatively late stage (approximately 15 hours (97)) . 5.
Sole o£ lysosomes (32) :
The case of the intersegmental muscles can
serve as a foil against which to discuss other phenomena .
In this tissue,
lysosomes are prominent and active in involution, but they are highly selec tive, destroying only mitochondria and ribosomes.
Such selectivity had been
noted previously (62, 63) and more dramatically in the skipper moth Calpodes, in which at different and sharply delimited times mitochondria and microbodies are removed by autophagic processes (90, 91) .
In degenerating epi-
thelial tissues, such as mammary tissue (38), insect salivary, silk, and prothoracic glands (7-9), neural tissue (48, 112) and liver ( 79) development of autophagic vacuoles is normally a dominant aspect of the destruction of the cell, the bulk of the cell at some point being filled with lysosomes or lysosomal derivatives .
Even in these tissues, however, the intracellular
control of lysosomal behavior is unknown.
In many tissues which ultimately
involute, lysosomes are present and apparently active during non-catabolic states (37, 83, 93) .
When cell death is induced, either the behavior of
the lysosomes must change or a new generation of the organelles, with different characteristics, must appear .
The selectivity mentioned above
suggests that cell organelles evolve so as to invite lysosomal attack ; in other tissues,
subtle morphological changes (28, 29, 70, 71) point to
similar conclusions .
Activation of pre-existent lysosomal enzymes can
occur, in the salivary gland of Chironomus , in the presence of inhibitors of protein synthesis.
It is claimed for this system (25, 65) that the
lysosomes actually rupture (133)--an idea which has otherwise fallen out of vogue.
A possibly related observation is that the formation of auto-
phagic vacuoles and autolysosomes in liver and flounder kidney tubules requires energy (2, 73, 75) but not protein synthesis (26, 73, 75, 142-5) . The evidence for the intersegmental muscles, mentioned above, is not necessarily contradictory, in that the protective effect of actinomycin D and cycloheximide may reflect synthesis of antecedent or unrelated proteins .
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Programmed Cell Death
There is in any event no information as to the intracellular signalling mechanism--how the endocrine or other signal is communicated, and how it ultimately evokes a predominantly catabolic behavior . 6.
Non-lysosomal mechanisms :
In many situations, most particularly
those of atrophying vertebrate (23, 113,
114, 130, 131) and invertebrate
(3, 4,116,135,140) muscle, in glucocorticoid-induced lymphatic involution (101) and cell death in chick limb buds (70, 71) the presence of lysosomes has been documented but their role is equivocal .
Lysosomal evolution is
either extremely minor or recognizable only very late in the process of involution .
In many situations (Table 2) moderately abnormal-appearing cells
are phagocytosed= the macrophage-type call digests the other by means of lysosome-derived organelles, but the dying cell displays no such activity ( 5, 59, 70, 71) . There exists another major mechanism of involution, which consists of early nuclear shutdown, followed by condensation of the affected cell, which is ultimately pbagocytosed without apeptosis (69, 70, 71, 87 and Table 2) . One tissue of this type has been studied extensively, thymocytes in the presence of glucocorticoids, and the primary biochemical lesion here appears to be an induced (nuclear?) impermeability to glucose (108) which is itself mediated by the synthesis of RNA and protein .
The relation of this meta-
bolic change to the morphologic appearance has not been studied, and comparison to other systems is not yet possible . Many other types of involuting cells undergo a phase of fragmentation or apoptosis similar to that of the intersegmental muscles.
Although the
material in the cell fragments is somewhat degraded, Karr observes that, by structural criteria, the metabolism of the fragments appears to remain intact (no swelling, mitochondria normal)(78-82) .
Thus, a considerable
fraction of a dying cell would be digested not in situ but at a different locus in the body, and the question of lytic enzymes would to some extent became moot . Although the apparent pH inside autophagic vacuoles is probably approximately 5 and thus within the range of the pH optima of most of the Lysosomal enzymes, cytoplasmic pH is probably between 6 and 7 (105) .
In
these conditions it is difficult to envisage activity of the lysosooal protease : and it must be admitted that the nature of intracellular, extralysosomsl proteolysis is not known. the intersegmental muscles as :
The question can be clearly stated for
How can an acid protease, pH optimum 3 .9,
contained within a discrete organelle, digest proteins exterior to the organelle while cytoplasmic conditions remain stable enough for the muscle to be normally contractile?
There is in fact surprisingly little direct
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Programed Cell Death
documentation that cathepsin D or similar acid-proteasss effect the degradation of any muscle proteins (19, 51, 84, 85, 105) .
As yet unsubstantiated
speculation suggests that in the highly-specialized Diptera, cell proteins escape intact to be re-utilised elsewhere (18, 28) .
Such an hypothesis would
be inconsistent with the prevailing interpretation of muscle catabolism (50, 51, 77) but should not be ruled out a priori for such highly-evolved animals . In vivo experiments currently in progress utilising pepstatin (5, 158) indicate that the rate of resorption of degenerating muscle is far less depressed than inhibition of cathepsin by the drug would suggest.
Furthermore,
several enzymes and other proteins disappear from the intersegmental muscles at . varying rates (Schlichtig, Lockshin, and Beaulaton, in preparation) suggesting less restricting means of catabolism than purely lysosomal. All of these data indicate as-yet-unknown means of degrading structural proteins which have been more thoroughly studied in pathological situations . 7.
Comparison to pathological situations f
For several reasons this
discussion is basically limited to similarities between the intersagmantal muscles and some myopathies .
Karr has drawn other analogies ( 80) and it
should be evident that phenomena such as call-msdiated immune lysis (52,72, 134) and lethal genetic abnormalities (67) are markedly different from the controlled dismantling processes described above.
Although obviously
the signalling mechanisms initiating the various myopathological responses differ--they range from loss of neurotrophic support in denervation (24, 47, 54-58) and perhaps muscular dystrophy (40, 153) through hormone withdrawal (58) and complex, unknown signals relating to use and to nutritional- status (50)--the intracellular changes are quite similar.
For all of the several
situations there is morphological (106, 107, 113, 118,
130, 131) or bio-
chemical (2, 23, 50, 114, 153) evidence for an increase in lysosoml enzymes during the catabolic phase.
Nevertheless, erosion of myofilaments occurs
external to these organelles .
More specifically, the morphologic appearance
of denervated muscle in the wax moth Galleria resembles that of the same muscle degenerating at metamorphosis, the only significant difference being in time scale (116, 117) .
A picture similar to experimental chloroquine
myopathy can be induced in a few hours in intersegmental muscle by the use of the drug at ecdysis, but not nearly so quickly prior to this time (38, 64, and in preparation) .
All of these observations suggest that the under-
lying lytic mechanisms are similar, but that the rates differ (122, 146) . Under normal circumstances the various myofibrillar proteins turn over at differing rates (46, 89, 101), and autophagy does not account for a large fraction of turnover (351 .
In situations involving protein degradation but
not cell death, the rate of destruction of individual proteins depends upon
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Programmed Cell Death
the size and inherent stability of the polypeptide chain, metabolic considerations, and other regulatory factors as yet not understood (50,
132) .
In
denervation- or cortisol-induced atrophy--which only in the extreme involves cell death--the rate o£ degradation of the long-lived molecules increases (50) . The differing catabolic rates suggest non-lysosomal modes of digestion. Although the evidence at this point is incomplete, it seems highly probable that the rate of degradation of contractile proteins of the intersegmental muscles must increase after ecdysis, and many of the electron micrographs suggest differential digestion of the various components (97, 98) .
Such
comparisons are much less readily made for other tissues, although the morphologic appearances are sufficiently similar for various workers to compare their results. 8.
Susaarys
The concept of Programmed Cell Death has been applied
to events in embryology , ageing, teratology, and carcinogenesis .
Of these,
only reproductive and developmental instances have been truly demonstrated to be "programmed" ; the finite life span of diploid cells in culture is programmed in an entirely different sense.
In the more limited scope we
would consider the process to include several identifiable events, including arrival and interpretation of an essentially innocuous signal, followed by experimentally detectable intracellular evolution. systems (93, 125, 126,
by RNA and protein synthesis. reversible .
In the best-studied
151) the earlier steps have proved to be mediated In the earliest phases, the process is
Once the process of dying is under way, subtle cytoplasmic
changes, perhaps ultimately deriving from decreased or altered nuclear activity, occur, and these primary changes may lead to activation or release of lysosomal enzymes.
There is little evidence to support the concept of
primary lysosomal attack, and in several systems (tadpole tail, chick limb bud, thymocytes, Calliphora intersegmental muscle) the lysosomes reside in other cells.
In most situations of controlled cell death, a large amount
of cytoplasmic material is ultimately jettisoned, and it is not until after this point that the cell gives evidence of not being able to control its environment .
While it is perhaps overly ambitious to extrapolate the term
"programming" to diverse phenomena, the intra-cellular events ensuing from the reception and interpretation of a triggering signal resemble, on an amplified scale, processes which occur during leas drastic diminution .
No
major contradiction separates any known aspect of programmed cell death, in the sense described above, from other developmental processes, or the mechanisms of lysis from known means of catabolism and physiological regulation .
It is therefore reasonable to assume that the phenomenon represents
an exploitable amplification of the currently mysterious control of catabolism,
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155 7
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ACKNOWLEDGEMENTS--Much of the more recent research was carried out with the support of National Science Foundation Grant GB-36905 . As-yet-unpublished research by R.Schlichtig, J . Parkinson, and J . Bidlack is considered in the text, and E.F . Adolph, L. Miller, and R. Connect have provided helpful comments . K. Srokose has provided consistently excellent technical assistance .
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Life Sciences Vol. 15, pp . 1549-1565 Printed in the U.S .A .
MINIREVIEW PROCRAMIO;D CELL DEATH Richard A. Lockshin Department of Physiology, University Dentistry, Rochester, N.Y . 14642 and de Clermont, Centre Scientifique des
1.
Definitions :
and Jacques Beaulaton of Rochester School of Medicine and Dipartement do Zoologie, Universiti Ciseaux, 63170 Aubiàre, France .
The term Programmed Call Death refers to several phe-
nomena in development, ageing, and pathology:
a finite limitation on the
number of generations cultured mammalian cells undergo (61), loss of cells during ageing, either sporadically (11,20,110,121,139) or in major episodes (12-19,27) ; or the destruction of large numbers of calls at specific points
of development (86,155) such as the loss of neurons in ganglia as others form synaptic connections (1,88) .
Other uses of the term include the normal,
exaggerated, or unusual loss of already-formed cells in developmental mutants such as rumplessness and apterous in Drosoahila (21,22,124,159,160) ; lytic responses of cells to essentially non-pathologic or developmental stimuli (70,71) including the appearance or disappearance of hormones (41-45 ;60,66,68, 69,115,136,148,156) ; and processes of dying known to include a complex sequence of identified, ultimately irreversible steps (93,125,126) .
The
latter categories include a vast number of examples, the range of which has been admirably summarised by GlUcksmann (49) and most recently revie"d by Saunders (125) .
Although the term "programmed" should most logically be
reserved for those instances in which a sequence of steps has been demonstrated, adequate documentation exists only for a few instances, and most workers have extended the concept to include what more correctly would be described as Itphysiological cell death" .
By this term we refer to the collapse of specific
cells and tissues in the absence of obviously toxic stimuli .
Acutely-killed
cells undergo a markedly different type of death, referred to as coagulative necrosis (78,79), which includes osmotic collapse or other processes of immediate disorganisation.
The definition suggested could include keratinisa-
tion of epithelial cells and cell death during normal turnover processes-essentially a cellular equivalent of metabolic turnover .
Although these
phenomena are relevant and interesting in their own rights, they are sufficiently different from the common concept or inaccessible to study to 1549
1550
Vol. 15, No . 9
Programmed Cell Death
warrant exclusion from a short review .
We will therefore concentrate on the
massive and controllable instances of cell death during development--situations which are most amenable to study and which provide a vantage point from which to relate the events of programmed cell death to the more general question of call loss from genetic, pathological, and temporal causes . 2.
Signalling mechanisms :
The great bulk of research in this area
has been concerned with the identification of instances of cell death and recognition of the immediately antecedent extracellular or physiological triggers .
The triggers, or controls, can be roughly divided into two
categories on the basis of the location of the control mechanism. logical regions of cell death,
Embryo-
such as in sympathetic ganglia (1) and avian
and mammalian limbs (70,71,76,125) appear to be controlled by locally diffusing materials .
The chemical nature of two substances which PREVENT cell
death (nerve growth factor and the substance which protects the cells of the posterior necrotic zone in chicks) has been established (1,26) and similar factors have been hypothesized but not identified in such situations as the bidirectional communication between neurons and their target organs .
Whether
or not these controls are routinely supportive, the absence of the control leading to involution, has not been determined .
More massive instances of
cell death are frequently mediated by more conventional hormones .
Such situa-
tions range from the involution of the Mullerian ducts under the influence of testosterone and duct-organizing substance (17,74,127-9) to the cataclysmic events of amphibian or insect metamorphosis (68,149-151 ; Table 1) . Although the supportive role of nerve growth factor may ultimately prove to relate to nutrition, inasmuch as the compound bears structural and functional similarity to insulin (1) other endocrine or diffusing chemical sig nalling mechanisms are not clearly related to intermediary metabolism, except perhaps for the lympholytic effect of cortisol (109) .
Other known signals are
idiosyncratic in nature and are not predictable in phylogenetic or other terms . Thus, in amphibia, thyroxin promotes the destruction of the tail musculature while stimulating the growth of the legs (151) ; in different species of beetles, the gonadotrophic juvenile hormone may provoke the development or the destruction of flight muscles, depending on the biology of the insect in relation to its repr9duction (16,33,136) . sexual tissue .
Similar arguments obtain for secondary
It is evident that the extracellular signals represent
an exploitation of accessible and controllable information, and that there is probably no clear order or reason to the type of signal utilized (93,
123) .
Any generality which could be drawn would relate, as for any developmental event, to the mechanisms of timing, recognition and response, and evolutionary adaptation of the physiological control mechanism .
Vol . 15, No . 9 3.
Intracellular avenu e
Programmed Call Death
155 1
Despite the many efforts to recognise the
axtracallular or physiological controls, surprisingly little attention has been devoted to the intracellular response phase of cell death (15) .
By far
the major number of studies are either documentation that cell death has occurred or morphological observations of the dying calls (Table 2) .
Focus
on the intracellular changes has for evident reason been limited primarily to situations of massive involution, such as amphibian and insect metamorphosis (36, 90, 91, 93, 151) and the collapse of mem ary and uterine tissue upon withdrawal of the supporting steroids (38, 156) .
Nevertheless, from thane
studies, several generalisations my be drawn, a)
Cell death normally affects specific cells and tissues, leaving
intact other, sometimes similar calls in the immediate vicinity .
Thus it
is unlikely that an extrinsic toxic substance provokes the collapse of the calls . b)
The dying cells themselves undergo considerable evolution prior to
a stage in which they lose control over their intracellular environment and amll or disintegrate (15, 142-145) .
Trump has underscored the major differ
ences between cells dying of insult such as anoxia and cells-falling prey to more biological phenomena (142-145) .
Karma, in emphasising the univer-
sality of a phenomenon he terms "apoptosis" (essentially a form of fragmentation of a dying cell) in a wide range of developing, involuting, malignant, and non-lethal pathologic situations, comments that apoptosis may be "possibly the only mode of controlled call death" (82) .
Although his generalisation
appears to be a bit too broad, the distinction is probably valid and forms the basis for the second generalisations
that there is no evidence that cells
dying in non-pathologic situations are suddenly cut off from vital supplies . Their evolution rather suggests that the intracellular milieu, as well as the extracellular, is under control and that the calls, although beating a retreat, are doing so in an organised and physiological manner .
There is
in fact some direct documentation for this statement, as is discussed below in section 4 and (103) . c)
Many forms of control, where studied, involve the synthesis of
RNA and protein prior to the beginning of involution .
Thus, antimetabolites
interfere with the collapse o£ tadpole tail tissues (140, 151), insect muscles (92), and thymocytes treated with glucocorticoids (108) .
Blockage
of protein synthesis does not appear to interfere with the activation of lytic (lysosomal) enzymes (25, 65) although the appearance of primary lysosomes may be blocked, as is discussed in section 4 below .
In the case
of thymocytes exposed to cortisol, the newly-synthesized protein say interfere with glucose uptake (108) ; elsewhere, the function of protein synths-
1552
Programmed Cell Death
Vol . 15, No . 9
sis is not known. d)
In a fear instances (12-14, 34, 66, 136,
nuclear lesions are reported .
147, 152) various primary
In this form of cell death, which is not
the most common, cell collapse without marked lysosomal involvement is normally followed by phagocytosis .
In other systems, the nucleus appears
to remain at least morphologically normal well into the lytic phase (Table 2) . e)
A fifth generalization is that the primary intracellular changes
which switch the cell to a catabolic state are in no sense known= nor is it certain what enzymes mediate the catabolism .
This statement is inten-
tionally and perhaps overly provocative, but is intended to counter an impression which pervades the literature on this subject .
In most but not
all tissues studied in any detail, the presence of an evolving lysosomal system
has been sought and found, and the lysosomes therefore invoked to
explain the tissue degradation .
Nevertheless, in many tissues primary changes
occur in the cytoplasm and these are on occasion flagrantly lytic (Table 2) . None of these changes have as yet been adequately explained . The significance, import, and limitations in these generalizations can be most effectively considered by examining a specific instance of cell death. Such an example could be the intersegmental muscles of silkmoths, for which a relatively broad range of information is now available (Table 3) . 4.
Illustrations
the intersegmental muscless
The intersegmental muscles
are larval muscles which are retained through the pupal stage until the emergence (ecdysis) of the adult insect, whereupon they begin to degenerate . These muscles are almost totally resorbed within 48 hours.
The signalling
mechanism involves an endocrine step at the outset of development which potenThree weeks later, ecdysis is
tiates or sets the stage for a future response .
initiated by a neuroendocrine signal (141) which activates a specific behavioral pattern including first hyperactivity and then quiescence of the intersegmental muscles (94) .
Same aspect of this change in motor activity appears to be the
proximate signal to initiate lysis (Table 3) .
Thereupon follows a period
involving the synthesis of new RNA and protein (92, 98) and the appearance in the tissue of large numbers of lysosomes (97) . are detectable biochemically slightly earlier (99) .
The lysosomal enzymes Large-scale breakdown
of the myofilamenta is recognized at an ultrastructural level within seven hours .
Lysosomes seem to be preoccupied with the destruction of mitochondria
and do not include myofilaments in their matrices .
During this phase input
resistance gradually increases and acme fragility (marked by a drop in capacitance when the fiber is placed in Ringo* appears (95, 96) but otherwise active and passive properties of the call membrane are normal . ating fiber is contractile .
The degener-
Approximately 12 hours after the emergence of
Vol . 15, No . 9
1553
Programmed Cell Death
the animal, autophagic vacuoles are seen in the tissue, though again they do not include myofilaments, and a phase of apoptosis or fragmentation is initiated.
It is only after this latter phase that the membrane potential
collapses.
Muscle proteins can be detected in the blood as early as three
hours after ecdysis, though they reach a peak during the height of fragmentation and collapse of the membrane potential,
24 hours after ecdysis (95) .
By 40
hours, only pycnotic nuclei and wisps of cytoplasm are left in the cell . Thus the breakdown of the intersegmental muscles involves the reception of two signals, synthesis of an unidentified protein, and activation of lysosomal and non-lysosomal lytic mechanisms .
Morphologic signs of nuclear
failure occur only at a relatively late stage (approximately 15 hours (97)) . 5.
Sole o£ lysosomes (32) :
The case of the intersegmental muscles can
serve as a foil against which to discuss other phenomena .
In this tissue,
lysosomes are prominent and active in involution, but they are highly selec tive, destroying only mitochondria and ribosomes.
Such selectivity had been
noted previously (62, 63) and more dramatically in the skipper moth Calpodes, in which at different and sharply delimited times mitochondria and microbodies are removed by autophagic processes (90, 91) .
In degenerating epi-
thelial tissues, such as mammary tissue (38), insect salivary, silk, and prothoracic glands (7-9), neural tissue (48, 112) and liver ( 79) development of autophagic vacuoles is normally a dominant aspect of the destruction of the cell, the bulk of the cell at some point being filled with lysosomes or lysosomal derivatives .
Even in these tissues, however, the intracellular
control of lysosomal behavior is unknown.
In many tissues which ultimately
involute, lysosomes are present and apparently active during non-catabolic states (37, 83, 93) .
When cell death is induced, either the behavior of
the lysosomes must change or a new generation of the organelles, with different characteristics, must appear .
The selectivity mentioned above
suggests that cell organelles evolve so as to invite lysosomal attack ; in other tissues,
subtle morphological changes (28, 29, 70, 71) point to
similar conclusions .
Activation of pre-existent lysosomal enzymes can
occur, in the salivary gland of Chironomus , in the presence of inhibitors of protein synthesis.
It is claimed for this system (25, 65) that the
lysosomes actually rupture (133)--an idea which has otherwise fallen out of vogue.
A possibly related observation is that the formation of auto-
phagic vacuoles and autolysosomes in liver and flounder kidney tubules requires energy (2, 73, 75) but not protein synthesis (26, 73, 75, 142-5) . The evidence for the intersegmental muscles, mentioned above, is not necessarily contradictory, in that the protective effect of actinomycin D and cycloheximide may reflect synthesis of antecedent or unrelated proteins .
1554
Vol . 15, No . 9
Programmed Cell Death
There is in any event no information as to the intracellular signalling mechanism--how the endocrine or other signal is communicated, and how it ultimately evokes a predominantly catabolic behavior . 6.
Non-lysosomal mechanisms :
In many situations, most particularly
those of atrophying vertebrate (23, 113,
114, 130, 131) and invertebrate
(3, 4,116,135,140) muscle, in glucocorticoid-induced lymphatic involution (101) and cell death in chick limb buds (70, 71) the presence of lysosomes has been documented but their role is equivocal .
Lysosomal evolution is
either extremely minor or recognizable only very late in the process of involution .
In many situations (Table 2) moderately abnormal-appearing cells
are phagocytosed= the macrophage-type call digests the other by means of lysosome-derived organelles, but the dying cell displays no such activity ( 5, 59, 70, 71) . There exists another major mechanism of involution, which consists of early nuclear shutdown, followed by condensation of the affected cell, which is ultimately pbagocytosed without apeptosis (69, 70, 71, 87 and Table 2) . One tissue of this type has been studied extensively, thymocytes in the presence of glucocorticoids, and the primary biochemical lesion here appears to be an induced (nuclear?) impermeability to glucose (108) which is itself mediated by the synthesis of RNA and protein .
The relation of this meta-
bolic change to the morphologic appearance has not been studied, and comparison to other systems is not yet possible . Many other types of involuting cells undergo a phase of fragmentation or apoptosis similar to that of the intersegmental muscles.
Although the
material in the cell fragments is somewhat degraded, Karr observes that, by structural criteria, the metabolism of the fragments appears to remain intact (no swelling, mitochondria normal)(78-82) .
Thus, a considerable
fraction of a dying cell would be digested not in situ but at a different locus in the body, and the question of lytic enzymes would to some extent became moot . Although the apparent pH inside autophagic vacuoles is probably approximately 5 and thus within the range of the pH optima of most of the Lysosomal enzymes, cytoplasmic pH is probably between 6 and 7 (105) .
In
these conditions it is difficult to envisage activity of the lysosooal protease : and it must be admitted that the nature of intracellular, extralysosomsl proteolysis is not known. the intersegmental muscles as :
The question can be clearly stated for
How can an acid protease, pH optimum 3 .9,
contained within a discrete organelle, digest proteins exterior to the organelle while cytoplasmic conditions remain stable enough for the muscle to be normally contractile?
There is in fact surprisingly little direct
Vol. 15, No . 9
1555
Programed Cell Death
documentation that cathepsin D or similar acid-proteasss effect the degradation of any muscle proteins (19, 51, 84, 85, 105) .
As yet unsubstantiated
speculation suggests that in the highly-specialized Diptera, cell proteins escape intact to be re-utilised elsewhere (18, 28) .
Such an hypothesis would
be inconsistent with the prevailing interpretation of muscle catabolism (50, 51, 77) but should not be ruled out a priori for such highly-evolved animals . In vivo experiments currently in progress utilising pepstatin (5, 158) indicate that the rate of resorption of degenerating muscle is far less depressed than inhibition of cathepsin by the drug would suggest.
Furthermore,
several enzymes and other proteins disappear from the intersegmental muscles at . varying rates (Schlichtig, Lockshin, and Beaulaton, in preparation) suggesting less restricting means of catabolism than purely lysosomal. All of these data indicate as-yet-unknown means of degrading structural proteins which have been more thoroughly studied in pathological situations . 7.
Comparison to pathological situations f
For several reasons this
discussion is basically limited to similarities between the intersagmantal muscles and some myopathies .
Karr has drawn other analogies ( 80) and it
should be evident that phenomena such as call-msdiated immune lysis (52,72, 134) and lethal genetic abnormalities (67) are markedly different from the controlled dismantling processes described above.
Although obviously
the signalling mechanisms initiating the various myopathological responses differ--they range from loss of neurotrophic support in denervation (24, 47, 54-58) and perhaps muscular dystrophy (40, 153) through hormone withdrawal (58) and complex, unknown signals relating to use and to nutritional- status (50)--the intracellular changes are quite similar.
For all of the several
situations there is morphological (106, 107, 113, 118,
130, 131) or bio-
chemical (2, 23, 50, 114, 153) evidence for an increase in lysosoml enzymes during the catabolic phase.
Nevertheless, erosion of myofilaments occurs
external to these organelles .
More specifically, the morphologic appearance
of denervated muscle in the wax moth Galleria resembles that of the same muscle degenerating at metamorphosis, the only significant difference being in time scale (116, 117) .
A picture similar to experimental chloroquine
myopathy can be induced in a few hours in intersegmental muscle by the use of the drug at ecdysis, but not nearly so quickly prior to this time (38, 64, and in preparation) .
All of these observations suggest that the under-
lying lytic mechanisms are similar, but that the rates differ (122, 146) . Under normal circumstances the various myofibrillar proteins turn over at differing rates (46, 89, 101), and autophagy does not account for a large fraction of turnover (351 .
In situations involving protein degradation but
not cell death, the rate of destruction of individual proteins depends upon
1556
Vol. 15, No . 9
Programmed Cell Death
the size and inherent stability of the polypeptide chain, metabolic considerations, and other regulatory factors as yet not understood (50,
132) .
In
denervation- or cortisol-induced atrophy--which only in the extreme involves cell death--the rate o£ degradation of the long-lived molecules increases (50) . The differing catabolic rates suggest non-lysosomal modes of digestion. Although the evidence at this point is incomplete, it seems highly probable that the rate of degradation of contractile proteins of the intersegmental muscles must increase after ecdysis, and many of the electron micrographs suggest differential digestion of the various components (97, 98) .
Such
comparisons are much less readily made for other tissues, although the morphologic appearances are sufficiently similar for various workers to compare their results. 8.
Susaarys
The concept of Programmed Cell Death has been applied
to events in embryology , ageing, teratology, and carcinogenesis .
Of these,
only reproductive and developmental instances have been truly demonstrated to be "programmed" ; the finite life span of diploid cells in culture is programmed in an entirely different sense.
In the more limited scope we
would consider the process to include several identifiable events, including arrival and interpretation of an essentially innocuous signal, followed by experimentally detectable intracellular evolution. systems (93, 125, 126,
by RNA and protein synthesis. reversible .
In the best-studied
151) the earlier steps have proved to be mediated In the earliest phases, the process is
Once the process of dying is under way, subtle cytoplasmic
changes, perhaps ultimately deriving from decreased or altered nuclear activity, occur, and these primary changes may lead to activation or release of lysosomal enzymes.
There is little evidence to support the concept of
primary lysosomal attack, and in several systems (tadpole tail, chick limb bud, thymocytes, Calliphora intersegmental muscle) the lysosomes reside in other cells.
In most situations of controlled cell death, a large amount
of cytoplasmic material is ultimately jettisoned, and it is not until after this point that the cell gives evidence of not being able to control its environment .
While it is perhaps overly ambitious to extrapolate the term
"programming" to diverse phenomena, the intra-cellular events ensuing from the reception and interpretation of a triggering signal resemble, on an amplified scale, processes which occur during leas drastic diminution .
No
major contradiction separates any known aspect of programmed cell death, in the sense described above, from other developmental processes, or the mechanisms of lysis from known means of catabolism and physiological regulation .
It is therefore reasonable to assume that the phenomenon represents
an exploitable amplification of the currently mysterious control of catabolism,
Programmed Cell Death
Vol . 15, No . 9
155 7
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ACKNOWLEDGEMENTS--Much of the more recent research was carried out with the support of National Science Foundation Grant GB-36905 . As-yet-unpublished research by R.Schlichtig, J . Parkinson, and J . Bidlack is considered in the text, and E.F . Adolph, L. Miller, and R. Connect have provided helpful comments . K. Srokose has provided consistently excellent technical assistance .
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