IV. RESULTADOS Y DISCUSIÓN
4.1. Tipos de relación familiar y actitud de los pacientes con TBC del Centro
The finding that over-expression of human annexin V in DT40s is lethal (section 5.8), is however of interest in itself. This result may also be related to the finding that micro-injection o f purified annexin V, but not annexin 1, into HeLa cells resulted in ceU- death over a period of 5 to 10 hours (H. Haigler, personal communication). This observation suggests that the phenomenon described in DT40 cells may also be true to lesser or greater degrees for other cell types, and is directly related to annexin V activity. However not all expression of annexin V can be lethal since some cell types, such as muscle (Giambanco et al., 1991), have considerable levels of annexin V expression without suffering cell-death. Perhaps in such cells the localisation or control o f annexin V function prevents lethahty.
Intriguingly H. Haigler also reports that electron-microscopy o f injected cells revealed that it was the mitochondria that showed the first observable damage, seeming to suffer membrane breakdown before destruction of further cellular architecture and membrane blebbing occurred. This form o f damage is consistent with apoptosis, perhaps initiated by mitochondrial dysfunction, though investigation of further characteristics typical of apoptotic cells, such as loss of membrane asymmetry and degradation of nuclear DNA could confirm this.
In addition to a lethal effect of over-expression of annexin V, the loss of expression of the endogenous gene results in an opposite resistance to apoptosis (section 5.9). It may be this resistance to apoptosis that results in a reduced background level of apoptosis in annexin V -/- clones o f 5 to 10% compared to wild-type cells (data not shown), leading to the observed increased growth rate (figure 5.1). However recent experiments suggest that annexin II -/- cells grown in the absence o f P-Me, also show increased growth rates and reduced levels of background apoptosis, suggesting that this phenomenon is not specific to annexin V, and that reduced background levels of apoptosis are in fact not related to the resistance to apoptotic stimuli shown by annexin V -/- clones.
To date the only link between annexin V and apoptosis was the use of fluorescently- labelled purified annexin V as a biochemical tool allowing detection o f the loss of plasma- membrane asymmetry that accompanies apoptosis in all cells; annexin V, like all annexins, by virtue of its abihty to bind negatively-charged phospholipids in the presence of calcium, labels such apoptotic cells (Reutehngsperger and van Heerde, 1997). Such an extracellular activity is unhkely to represent a biological function (section 1.1 1), however the results
described in section 5.9 strongly suggest that annexin V does have a specific biological function in apoptosis, though most likely as an intracellular protein involved in the control of the process.
Further evidence for a general role of annexin V in vertebrate apoptosis during both development and adult life comes from two sources. Firstly C. Reutehngsperger reports that developmental expression patterns of annexin V show significant overlap with tissues undergoing apoptotic cell death, such as the inter-digital tissue of the hands (personal communication). Secondly the drug K201, which may have annexin V as its target, is able to inhibit the cellular damage, thought to be apoptotic, that accompanies calcium overload in the heart (section 5.14). Furthermore, a general apoptotic role for annexin V could explain it’s possible tumour suppressor effects (Karube et al., 1995), shared by the related annexin VI, but not by annexin II, whose loss also has no effect on apoptosis (section 5.9).
While annexin V may thus be an important regulator of apoptosis, its mode of action is still unclear. Such a lack o f clarity of function is however common to many of the proteins, such as bcl- 2 family members, known to be involved in the apoptotic process
(Reed, 1997). The phenomenon of apoptosis has been reviewed extensively (Leist and Nicotera, 1997; McConkey and Orrenius, 1997), and while the regulation and execution of
the process is still far from clear, the salient points that are broadly accepted are as follows. The process involves the controlled degradation and packaging of cellular components for phagocytosis by neighbouring or scavenging cells, leading to cell death in a programmed manner that avoids inflammatory responses that could be damaging. This is of importance in the organism in development, for example in the brain, where excess neurones are produced and those that faü to make the correct connections commit apoptosis, in the immune system, where non-responsive or self-responsive T and B-cells are eliminated, and during the pathological damage following stroke in the brain, or infarction in the heart.
The signals that lead to apoptosis are various, from cytokines such as fasL and TNF, over-stimulation by for example the B-cell receptor, ischaemia, viral infection, and a broad range of cytotoxic agents from irradiation to drugs such as staurosporine (Steller, 1995). Likewise there are probably multiple cellular initiators o f apoptosis in response to these signals, which are for the most part unknown, except for the direct receptors for fasL and TNF (Nagata and Golstein, 1995; Smith et al., 1994). Furthermore these signals activate several pathways in the cell, some of which are of more importance in one cell type than another, and in response to one stimulus than another. The best characterised of these pathways are the caspase proteolytic cascade (Thomberry and Lazebnik, 1998), and the mitochondrial permeabihty transition and cytochrome C release (Green and Reed, 1998). These pathways are closely inter-linked, so that caspase activation promotes the mitochondrial permeability transition, and the resulting cytochrome C release enhances caspase activation, creating a degree of positive feed-back (Leist and Nicotera, 1997).
Members of the bcl-2 family both inhibit these pathways, such as bcl-2 itself, and promote apoptosis, such as bax. The site of action of bcl-2 family members in this pathway is unclear though, being possibly at the endoplasmic reticulum membrane, the mitochondria, or the cytosol. Furthermore the mode of action of members of the bcl-2 family is also unknown, possibly being related to their in vitro pH-sensitive pore-forming activities (Reed, 1997). Thus although no stmctural or sequence homologies have been identified to date, there are some functional similarities between bcl- 2 family members and
annexins such as annexin V; annexin V may also have various functions, including pro- apoptotic roles, at a range of intracellular membranes including the mitochondria, and has in vitro channel activity, perhaps also by self-insertion, possibly in response to pH (Kirsch et al., 1997; Kohler et al., 1997).
Downstream of the above positive feedback loop, many mechanisms are activated including loss o f plasma-membrane asymmetry, thought to be a signal for phagocytosis, organelle breakdown, nuclear condensation, perhaps as a result of nuclear laminin cleavage by caspases, and DNA fragmentation (Leist and Nicotera, 1997). Some, and generally all of these events may occur in an individual apoptotic cell, depending on the cell type and the apoptotic stimulus.
The treatment of a cell with a particularly acute stimulus may induce aspects of this program, though extreme damage may prevent its full execution, leading to uncontrolled cell lysis, often described as necrosis. Thus milder stimuli are likely to be more revealing of physiological apoptotic pathways. Of particular importance in the process of apoptotic decision-making may be the mitochondrial state; a cell with defective mitochondria will not be able to synthesise the ATP required to complete the energy-requiring apoptotic program. Thus an early sensor of cell damage may be the mitochondria; if damage is detected, apoptosis must be induced before ATP production fails (Leist and Nicotera, 1997). Should such an early-warning system fail, necrosis and accompanying inflammation are likely to ensue at a later time.
6 .6
Annexin V and calcium are involved in the same apoptotic pathways.
The results presented above indicate that loss of annexin V does not protect against all apoptotic stimuh (figure 5.12); UV-induced apoptosis is unaffected by the loss of annexin V. Indeed radiation-induced apoptosis in DT40 cells has been shown, by similar targeted disruption techniques, to be dependent on Bruton’s tyrosine kinase (btk) (Uckun et al., 1996). This implies that annexin V is not part of the pathway involving btk, but may be part o f an alternative apoptosis-inducing pathway activated by other cellular insults. Furthermore the observation that caspase 3 activation is greatly reduced in annexin V -/- cells (figure 5.13), also suggests that annexin V operates upstream of the central caspase/cytochrome C program.
Given that annexin V binds Ca^^-dependently to membranes, and may act as a Ca^^ channel, the role of calcium in the induction of apoptosis may be related to that o f annexin V; could annexin V respond to, or mediate changes in Ca^^-flux within the cell leading to apoptosis? Indeed UV-induced apoptosis is Ca^^-independent, while staurosporine and chelerythrine-induced apoptosis are Ca^^-dependent (figures 5.14 and 5.15). These stimuh thus show the same calcium and annexin V dependency. Furthermore the calcium requirement during apoptosis induced by staurosporine or chelerythrine is early on, before maximal caspase activation is achieved (figure 5.13), suggesting that, like annexin V, calcium is required early in these pathways, thus providing an additional temporal connection. These results are summarised in the model depicted in figure 6.1.
Evidence that calcium is important in the regulation of apoptosis, as suggested above in drug-induced DT40 apoptosis, is becoming clear from a wide range of experiments in many systems (McConkey and Orrenius, 1997; Nicotera and Orrenius,
1998). High levels o f intracellular calcium have long been known to lead to cell-damage and apoptosis, as shown in hypoxia-induced cardiac calcium overload (Halestrap et al., 1997), and by treatment o f lymphoid or prostate tumour cells with calcium ionophores (Kaiser and Edelman, 1978; Martikainen and Isaacs, 1990). Furthermore apoptotic stimuli
Figure 6.1 A model
the role of annexin V in apoptosis.
UV D ru gs e .g . S ta u ro sp o rin e
Ca2+
AnxV \ C a s p a s e a c tiv a ti o n \ M ito c h o n d ria l A A p o p to sissummarising
have also, like many other stimuli, beenshown to be associated with enhanced calcium influxes as shown with glucocorticoid-induced thymocyte apoptosis (Kaiser and Edelman, 1977), and staurosporine-induced apoptosis in PC 12 cells (Kruman et al., 1998).
Inhibition of such calcium influxes with Ca^^-channel blockers has also been reported by several groups to inhibit apoptosis in a range of systems in a similar way to that described for DT40 cells in section 5.11. Thus Ca^^-channel blockers also inhibit apoptosis induced by testosterone withdrawal in the regressing prostate (Martikainen and Isaacs, 1990). Furthermore blocking of calcium fluxes with intracellular Ca^^ buffering agents and extracellular Ca^^ chelators, can inhibit early events such as caspase activation, as well as DNA fragmentation and cell death (McConkey and Orrenius, 1996). Thus Ca^"^ may indeed be involved in early induction steps of apoptosis as the results in section 5.12 indicate.
How calcium could be involved in the regulation of apoptosis is however still an area of speculation. One interesting possibility is that the Ca^Vcalmodulin-dependent protein serine/threonine phosphatase, calcineurin, may regulate a calcium-dependent apoptotic step. Indeed cyclosporin A, an immunosuppressant that inhibits calcineurin, can block Ca^^- dependent apoptosis in lymphoid model systems (Amendola et al., 1994; Makrigiannis et al., 1994). Furthermore, recent work has shown that bcl-2 suppression of NF-AT (nuclear factor of activated T cells), which is activated during Ca^'"-dependent apoptosis, is linked to bcl-2 binding of calcineurin and inhibition of its function (Shibasaki et al., 1997).
Thus reduced calcineurin activity, perhaps leading to the abrogation of NF-AT activation, may be the cause of the reduced levels of apoptosis in annexin V -/- cells. Further experiments investigating the activation of calcineurin and NF-AT-dependent gene transcription during the induction of calcium-dependent apoptosis in DT40 cells will thus be of interest. A generally reduced basal activity of a protein serine/threonine phosphatase, such as calcineurin, could also explain the lack of homotypic adhesion in annexin V -/- cells as discussed in section 6.3, providing a possible link between these apparently divergent phenotypes.
Alternatively, annexin V -/- cells could be defective in the calcium fluxes that may activate such calcium-dependent pathways in apoptotic cells. This is particularly of interest since annexin V may act as a calcium channel, and the H2 0 2-induced calcium responses.
which lead to apoptosis (figure 5.12), are altered in annexin V -/- cells as discussed in section 6.4. However as shown in section 5.13, at least over the time-period examined, the cytosolic calcium responses to staurosporine, and probably chelerythrine, are normal in annexin V -/- cells. Although these results imply that annexin V is not acting as a channel during apoptosis, the results with K201 (section 5.14) may suggest otherwise. Thus K201, a potential annexin V channel blocker, does inhibit chelerythrine-induced apoptosis, and acts over a similar time-course to other inhibitors of Ca^^ flux, providing a strong circumstantial link between K201 binding of annexin V and inhibition of Ca^^ flux.
Preliminary experiments suggest however, that K201 may have no effect on staurosporine-induced apoptosis (data not shown), potentially confounding such a link. Future work will thus involve investigating the effect of K201 on induction of apoptosis by staurosporine and other agents. Additional preliminary experiments also suggest that K201 has no effect on homotypic adhesion, or H^O^-induced Ca^"^ entry (data not shown), again casting doubt on its ability to act as a true annexin V inhibitor. However it is possible that K201 only inhibits, for example, the proposed membrane embedding o f annexin V, an event perhaps only required during normal apoptosis, and not as part of the other annexin V-dependent events described above. Alternatively, only apoptosis may be directly dependent on annexin V channel activity and thus sensitive to K201. In either case further studies with K201 will be of interest to determine whether annexin V is its true biological target, and to investigate its possible effects in apoptosis and other cellular functions, given its role as an inhibitor o f myofibrillar over-contraction and associated damage (section 5.14).
Thus while there is circumstantial evidence for a link between annexin V and calcium flux in apoptosis, to some extent supported by the results with K201 discussed above, there is no direct evidence for changes in cytosolic calcium signals in response to apoptotic stimuli in annexin V -/-, compared to wild-type cells. There are several possible solutions to this paradox. Firstly annexin V may act independently of calcium, secondly annexin V may act downstream of calcium signals, perhaps in the calcineurin pathway described above. A third explanation could be that calcium signals dependent on annexin V may occur in cellular compartments other than the cytosol, and have thus not been detected in the experiments descibed in section 5.13. Finally any annexin V-dependent cytosolic apoptotic calcium signals may still occur, but after the 1 0 minute time-course investigated in
section 5.13.
Regarding the last of these possibilities, figure 5.15 suggests that changes in calcium flux are of most importance within the first 30 minutes to 1 hour after addition of a drug. Thus any differences in response that occurred between 10 minutes and 1 hour which may be o f significance, would not have been observed in figure 5.16. However the experimental set-up used in section 5.13 is not suitable for such long time-course experiments, due to baseline drift and cellular damage; single-cell fluorometric
measurements by microscopy over a longer time-course may thus have to be employed for such experiments. The results do show though, that while H2 0 2-induced calcium responses
may be abnormal in annexin V -/- cells, other apoptotic stimuli give normal responses over similar time-courses. It is thus not a general loss of late-phase calcium entry in response to apoptotic stimuli, as shown in response to H2O2 by annexin V -/- cells, that causes the
reduction in apoptosis in annexin V -/- cells.
A yet later phase Ca^"^ entry pathway operating between 10 and 60 minutes after drug treatment is thus still a possible site of action of annexin V as described above. However should no changes in global calcium signalling between wild-type and annexin V -/- cells be apparent in response to any apoptotic stimuli, a role for annexin V as a calcium channel in apoptosis still cannot be excluded; there are several ways in which a Ca^^ channel could affect apoptosis without necessarily affecting global cytosohc calcium. Thus, for example, increased locahsed channel activity at the plasma-membrane could have local signalling effects on Ca^^-sensitive pathways. Such influx could be counterbalanced by increased Ca^^-extrusion activity or Ca^^-uptake by intracellular compartments, thus maintaining low global cytosolic free calcium concentrations. Indeed locahsed Ca^^ entry has been reported in cells just beneath the plasma-membrane (Marsault et al., 1997).