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Join Dr Lisa Bouchier-Hayes as she discusses the imaging based tools developed to interrogate caspase pathways.
Hello and welcome to Abcam's webinar on Lighting up the pathways to cell death. Today's principal speaker is Lisa Bouchier-Hayes, Assistant Professor in the Department of Pediatrics and Molecular Cell Biology at Baylor College of Medicine in Houston, Texas. The focus of Lisa's research is to determine how many caspases function in apoptosis and non-apoptotic processes to protect from disease. Her group develops and uses imaging-based methodologies to interrogate activation of distinct caspases in normal and transformed cells, and how their activation impacts cell fate. A major focus of her lab is to investigate the role of caspase-2 as a tumor suppressor. Their overall goal is to determine how caspase pathways can be manipulated for preventative and therapeutic purposes.
Joining Lisa today will be David Bruce, Business Development Associate at Abcam. David has a BSc in Biomedical Sciences from the University of Aberdeen. Before joining Abcam, David studied for a PhD at the MRC Protein Phosphorylation Unit at the University of Dundee. He examined the regulation of the TGF beta signaling pathway by novel mechanisms. I will now hand over to Lisa who will now start this webinar. Lisa?
LB-H: Hi, and thank you Lucy for a very nice introduction, and thank you everyone for joining us for this webinar. So, as Lucy introduced, today I'm going to talk about imaging-based strategies that we have used to interrogate caspase function. So just as a brief headline to my talk today, I'm going to describe what caspases are and how they function in apoptosis. I'm also going to talk briefly about some emerging non-apoptotic roles for caspases. The main part of the talk, as I said, is how to describe imaging tools that we have used to measure caspase activation; and specifically how we've used these tools to interrogate the caspase-2 pathway and its role between the suppressors.
So the caspases are a family of proteases, and these are the human caspases in their schematic form. As you can see they all have very similar structures, they all contain, for the main, a large and a small catalytic subunit. Within the large catalytic subunit is this conserved motif QACRG, which contains an active site 15 which is required for the enzyme activity of these caspases. The caspases can be loosely divided into three groups based on their function, and starting from the bottom we have the executioner caspases, including caspase-3, 6 and 7. Next, we have the initiator caspases, including caspase-2, 8, 9 and 10, and as their names imply, the initiator caspases initiate the apoptotic signal, while the executioner caspases carry out the execution phase of apoptosis. The third group are the inflammatory caspases and these don't really function in apoptosis, they are more involved in inflammation signaling and they include caspase-1, 4 and 5. But, as you can see from this slide, the initiator caspases and the inflammatory caspases look very similar, they all have long prodomains that contain within them protein/protein interaction motifs shown in orange or purple. So these are CARD or DED factor domains, and these protein/protein interaction motifs are very important for the function of these caspases, which I'll go into in more detail later. So, in some ways, the inflammatory caspases can also be considered initiator caspases.
So what do caspases do? Well, they cleave substrates and they do so in a very specific way, and this is just a nomenclature we use to describe high caspases cleaved by substrates. So immediately upstream of the caspase cleavage site, the amino acid is termed the P1 position, and as we go back, it's called P2, P3, P4, etc. Immediately after the cut slide is termed the P1 prime position, and as we look at the preferred caspase substrate sequences for caspases-1 through 9, you'll notice that they are pretty well-conserved, so usually four or five amino acids long. Always in the P1 position you have an aspartate, so caspases cleave after aspartates.
There is a huge repertoire of caspase substrates and many of these are structural and regulatory proteins, and many of them have distinct roles in apoptosis. For example, caspases cleave other caspases and this serves to amplify the caspase cascade, committing the cell to apoptosis. Many of the changes in the nucleus that are hallmarks of apoptosis are due to cleavage of substrates. For example, cleavage of iCAD contributes to DNA fragmentation, acinus cleavage contributes to chromatin condensation and cleavage of lamins lead to disruption of the nuclear envelope. Similarly, changes of plasma membrane can be attributed to cleavage of certain substrates, so cleavage of gelsolin and ROCK-1 kinase, and p21-activated kinase have been shown to contribute to the membrane blebbing that we see as the cell undergoes apoptosis. Finally, caspases cleave many regulatory proteins to disrupt organelle functions, so, for example, in the mitochondria NDUFS1 is a crucial component of the electron transport chain. This is cleaved by caspases, and lead to disruption of the electron transport and, hence, disruption of mitochondrial function. So there are a number of substrates of caspases that get cleaved during apoptosis. What's important to know that there hasn't been one substrate identified that is solely responsible for cell deaths. So it seems to be more a combination of a large number of substrate cleavage events.
So the caspases work together to induce apoptosis, and the main pathway that does so is the mitochondrial or the intrinsic pathway. So stimuli such as DNA damage, ER stress or metabolic stress lead to permeabilization of the outer mitochondrial membrane, and this is a step we call MOMP for short. Upon MOMP, cytochrome c that normally resides in the intermembrane space of the mitochondria, gets released from the mitochondria. Once it's released into the cytosol it leads to the activation of the initiator caspase, caspase-9, which in turn cleaves and activates the executioner caspases, caspases-3 and 7. The executioner caspases have a much larger repertoire of substrates than the initiator caspases, so these cleave multiple structural and regulatory proteins throughout the cell, including the one they listed, and to bring about the death of the cell. The second major pathway is known as the extrinsic pathway, or the death receptor pathway and the ligation of death receptors of the cell membrane leads to activation of the initiator caspase, caspase-8. Which in turn cleaves and activates the executioner caspases, caspase-3 and 7. In some cell types caspase-8 can feed into the mitochondria pathway, and lead to activation of the downstream caspases in a more indirect fashion.
So the main difference between the executioner caspases and the initiator caspases is how they are activated. The executioner caspases that are present in the cell as preformed dimers, and they are activated by cleavage, so they are cleaved between the large and small subunits to form the active enzyme. In contrast, the initiator caspases are present in the cell as inactive monomers, and they are activated by dimerization. This dimerization occurs when the initiator caspase monomers are recruited to large molecular-like complexes and proteins that we loosely term activation platforms. So the activation platforms act as molecular scaffolds for the initiator caspases, recruiting these monomers and bringing the monomers close together in a process we term 'induced proximity'. Induced proximity allows for dimerization of initiator caspases, and at this stage is considered active. Once it is dimerized the caspase undergoes auto cleavage, and this serves to stabilize the active enzyme. So the take-home message from this slide is that for initiator caspases, cleavage is not sufficient for activation, it must be dimerized first in order to be activated. In fact, it has been shown for many of these caspases that if you cleave these caspases in the absence of dimerization, you will not get any activity.
So recruitment to these activation platforms is mediated by protein/protein interaction domains, such as the CARD domain shown here. There's a number of these protein/protein interaction domains have been characterized and collectively they're called death fold protein/protein interaction domains. So these include the death effector domain, the caspase recruitment domain or CARD, both of which are present in some of the initiator caspases that I've shown you. There's also a related domain called the death domain, and this is found in a number of proteins that make up activation platforms. These domains mediate specific protein/protein interactions that regulate activation platform assembly.
So a number of different activation platforms exist, and they are each assembled to recruit specific initiator caspases. So the most well-documented of these is probably the death-inducing signaling complex, or the DISC which is assembled in response to death ligands such as Fas ligand. Assembly of the DISC leads to recruitment and activation of caspase-8. Similarly, the Apaf-1 apoptosome is assembled in response to MOMP and cytochrome c release, and this leads to the recruitment and activation of caspase-9. Caspase-1 is one of the inflammatory caspases and although it doesn't primarily induce apoptosis, it is also activated in a similar way by recruitment to a complex term the 'inflammasome'. Finally, caspase-2 appears to be activated on recruitment to a complex known as the 'piddosome'.
So caspases are essential for inducing apoptosis, but even when caspases are not activated in some cases the cell will still undergo a form of cell death. This occurs if caspases are blocked downstream with the mitochondria, the presence of cytochrome c and the destruction of the mitochondrial membrane will still lead to a caspase independent cell death. So the cell will eventually die, and this is what it looks like, so this is classical apoptosis where the cell shrinks and it starts to bleb. If we inhibit caspases under the same treatment conditions, you see the cells look roughly the same as in the control situation, but these cells will very rarely grow out and they will die by caspase independent cell death.
In the last number of years, it has emerged that caspases not only function in apoptosis, but they also have non-apoptotic roles. Some examples of these are in proliferation, so caspase-8 is required for the proliferation of T, B and NK cells. Conversely, caspase-3 has been suggested to inhibit B cell proliferation, and caspase-3, 8 and 14 have been shown to have roles in senescence and differentiation. The inflammatory caspases are primarily involved in inflammation and they do so by processing and maturing pro-inflammatory cytokines, and some caspases have also been shown to activate NFkB. Finally, some caspases may have a cell survival function, in particular caspase-8 appears to inhibit a different form of cell death known as necroptosis or programmed necrosis.
So just a couple of examples of these, and caspase-1 which is one of the inflammatory caspases, its main function is to cleave pro-inflammatory cytokines, including proIL-1 beta. This is just a simple schematic of the pathway where bacterial pathogens are engaged to toll-like receptors of the cell membrane, and this leads to activation of NFkB. One of the targets of NFkB is proIL-1 beta and you can see this in the western blot in THP-1 monocyte cells. Treatments with LPS after two hours leads to profound induction of proIL-1 beta expression.
At the same time, caspase-1 is activated and although on the western you can't really see any difference in the appearance of caspase-1; caspase-1 does go on to cleave proIL-1 beta to its mature form IL-1 beta, which is released from the cell and you can measure this by ELISA. The IL-1 beta as a number of roles in pathogen clearance, and this pathway is important for protection from sepsis and other inflammatory disorders.
Caspase-8, as I said, seems to have a pro-survival function in certain contexts by inhibiting another form of cell death called necroptosis. So caspase-8 is activated upon recruitment to death receptors, so death receptors recruit a complex including FADD and caspase-8, but it also engages the secondary complex made up of RIP kinase 1 and RIP kinase 3. Under normal circumstances, caspase-8 maintains this complex inactive to prevent necrosis, but in mice where caspase-8 has been knocked out this complex leads to uncontrolled necrosis, and embryonic death. If we look at mice that have knocked out both caspase-8 and RIP kinase 3, this restores embryonic development, but as the mice are born they eventually get T-cells with proliferation defects. So caspase-8 appears to be important, not just for inducing apoptosis, but for keeping the necroptosis pathway in check in preventing uncontrolled death from the RIP kinase proteins.
So how do we measure caspase activation? Well, the most popular way is to look at cleavage caspases by western blot. So, for example, in the western here treating HeLa cells with actinomycine d leads to cleavage in caspase-3. We see disappearance of the full-length and appearance of two bands corresponding to the large and the small subunits. You can also probe these blocks with an anti-active caspase-3 antibody, and get a similar band indicating the active form. If we co-treat with a caspase inhibitor such as QVD, it completely inhibits the cleavage of caspase-3.
So while this is a really good way of looking at activation of executioner caspases, as we've discussed, the initiator caspases do not require cleavage to be activated and they are activated by dimerization. So this can lead to some problems when using these kind of approaches to look at activation of initiator caspases. So, for example, in this experiment we do see cleavage of caspase-2 in response to actinomycine d. We see disappearance of this 15 KD proform, and you can just about make out the appearance of the cleavage product around 15 KD. So although it is cleaved, this is not necessarily evidence that it has been activated. So these kind of approaches need to be controlled very well and treated, and interpreted correctly.
Similarly, we often use cleavage fluorescent caspase substrate, so these substrates comprise the preferred caspase substrate motif, such as DEVD or VDVAD and that is attached to a fluorogenic moiety so that when they get cleaved it becomes fluorescent. So many of these substrates have overlapping specificities, so although they're good at telling us overall caspase activity, they are less good at telling us which specific caspase has been activated.
So for these reasons, we wanted to see if we could develop ways, new ways of looking at caspase activation and, in particular, initiator caspase activation. We also wanted to develop ways that we could do this by using imaging techniques, and time-lapse confocal microscopy. The reason for this is that time-lapse confocal microscopy can tell us a lot about what's happening during apoptosis in single cells at particular time points, that can often be missed when looking at cell populations.
So I'm going to show you an example of this in a movie in the next slide, but I'm just going to go through the steps of what you're going to be looking at first. So these cells are HeLa cells that stably express cytochrome c-GFP, which you can see in green, and these localize to the mitochondria. They've also been stained with a dye called TMRE, which binds to mitochondria which has high transmembrane potential. So when the cells are treated with actinomycine d, they undergo apoptosis. Two things happen initially: first, cytochrome c is released, so it goes from the mitochondrial punctate distribution to a cytosolic distribution where it is diffused throughout the cell. Around the same time, the mitochondria lose their transmembrane potential, so we lose the red dye. Then shortly after this, caspases get activated and we see shrinkage and membrane blebbing, which are some of the hallmarks of apoptosis. We also observe caspase-dependent changes at the cell membrane, and one of these is phosphatidylserine flip; so the lipid phosphatidylserine moves from the inner-leaflet of the plasma membrane to the outer-leaflet of the plasma membrane. We can detect this with an exon 5, which binds to the phosphatidylserine and this is shown in blue. Finally, at least in culture, the cell membrane ruptures and we can determine this using cell impermeable dyes that upon rupture the membrane will go into the cell. The dye we use here is propidium iodide, which binds to the nucleus, so we see the nucleus lighting up in pink.
So if we put this together into the movie we see cytochrome c released, loss of TMRE, cell shrinkage, blebbing and exon 5 binding, and PI uptake. So we can measure multiple events during apoptosis at the same time, and this is a really useful way of pinpointing when and how things occur. But in this movie there isn't a direct measure of caspase activation.
So there are a couple of ways of looking at caspase activation in live cells using microscopy techniques. One of these is to use FRET-based caspase substrates, so FRET stands for Fluorescence Resonance Energy Transfer, and this uses pairs of fluorescent proteins such as CFP and YFP, and CFP, in this case, acts as a donor and YFP acts as an acceptor. We link the CFP and YFP by a caspase cleavage slice such as DEVD, and when these are close together you can excite CFP and it will donate a duct of energy to the acceptor YFP and you can measure the level of FRET by the amount of emission from YFP. So when these are together the FRET levels are high. When caspases are activated they will cleave this probe and the CFP and YFP will disassociate from each other, and this will lead to a disruption FRET level, so FRET levels will be low. So these FRET probes can be used to measure caspase activity in live cells.
So here's an example, which was provided for us by our collaborator, Markus Rehm, and in the images above the cells were stained with TMRM, which like TMRE, binds to the mitochondria with high transmembrane potential. At the same time as cytochrome c release, we see loss of TMRE at this 318 time point. In the lower panel we're looking at the cleavage of the FRET probe, so when the probe is intact the colors you see are cooler, so blues and greens, but as it moves to warmer colors, orange and reds, this indicates cleavage of the probe. So you'll notice that we start to see the orange color appearing, indicating probe cleavage around the same time as we see loss of TMRE staining.
In the lower graph, the line trace shows a cell that has been treated with TNF, which engages the death receptor pathway. By conferring a FRET probe cleavage to events such as MOMP in the cell, or loss of TMRE we can infer which caspase has been activated. So in this example, there is a small amount of FRET probe cleavage prior to MOMP, which can be attributed to either caspase-8 or 10. Then shortly after MOMP there's a huge burst of cleavage, which can be attributed to the executioner caspases.
So, more recently, we wanted to find a more direct way of looking at initiator caspase activation. To do this we adopted a technique called bimolecular fluorescence complementation hybridization. This uses split fluorescent proteins such as VENUS, which is a brighter and more photostable version of YFP. When it is split into two fragments, so fragments on their own are not fluorescent, but when they are brought back together they become fluorescent again. So this can be used to look at proteins binding to each other, so as the proteins A and B bind it forces the two halves of VENUS to refold and become fluorescent.
So we wanted to use this to look at initiator caspase activation, and in particular we looked at the caspase-2 pathway. So just as a reminder, caspase-2 is an initiator caspase, like the other initiators it has a long prodomain which contains a protein/protein interaction motif: in this case a CARD domain. It is present in the cells in active monomers and gets activated upon recruitment to its activation platform. The activation platform for caspase-2 appears to be a complex termed the PIDDOSOME comprising of the proteins PIDD and RAIDD. RAIDD contains a CARD domain which binds to the CARD in caspase-2, leading to induced proximity of caspase-2 monomers, dimerization and activation. So given that recruitment of caspase-2 to its activation platform is the first step in activation of this caspase, we wondered if we could use the BiFC technique to look at caspase-2 induced proximity and dimerization.
So to do this we took the N terminus of VENUS and fused it to the CARD domain of caspase-2, so this is the portion of caspase-2 that binds to RAIDD. We also took the C terminus of VENUS and also fused that to the CARD domain of caspase-2. Such that when caspase-2 gets recruited to its activation platform, the two halves of VENUS come together and lyse up.
So it’s proof of principle we express the caspase-2 CARD BiFC pair in cells with components of the PIDDOSOME, PIDD or RAIDD. So in the presence of PIDD we saw about 40% of the cells became fluorescent, and in the images the red is DsRed-Mito which is a reporter for transfection, all of these experiments were done by transient transfection. So it's just to show that the cells are transfected, and so we can also localize the cells. When we expressed RAIDD, which is the direct adaptor for caspase-2, the majority of the cells became positive for caspase-2 BiFC. As negative controls were used to Apaf-1, which is a CARD-containing protein, and FADD which is a death domain-containing protein. So this indicated that we could reconstitute the PIDDOSOME to induce caspase-2 BiFC, so it looks like a good measure of caspase-2 activation.
Then we wanted to see if we could see caspase-2 BiFC after activation of endogenous receptors. So we used heat shock, which is a stimulus we knew could activate caspase-2, and in this graph we titrated the amount of the caspase-2 CARD pair, and even at the highest levels there wasn't a huge amount of background fluorescence. When we heat shocked the cells there was a very specific induction of caspase-2 BiFC, and this was what the cells generally look like. We saw a series of small spots of complexes and in green representing caspase-2 fluorescence complementation, in a perinuclear fashion.
We looked at other inducers of apoptosis, first anti-Fas cycloheximide, which doesn't seem to activate caspase-2 and this experiment doesn't induce any caspase-2 BiFC. Treatment with TNF cycloheximide induced a small amount of caspase-2 BiFC, there was about 20% of the cells with minimal brightness in each cell. Similarly, treatment with the DNA damaging agent etoposide also induced caspase-2 BiFC in about 20% of the cells, with minimal brightness in each cell. We saw much more efficient results with taxol and vincristine, which gave substantial induction of caspase-2 BiFC; and these are both cytoskeletal disruptors.
So what's really nice about this technique is we can look directly at the kinetics of caspase-2 activation in real-time. So, again, the red here are as a mitochondria showing the cell has taken up the plasmid, and we treat this cell with heat shock and we see over time the appearance of these green spots, which represent caspase-2 being recruited to its activation platform, undergoing induced proximity and dimerization.
We can express this graphically to determine exactly when this process starts, so after heat shocking the cells at 45°C for 1 hr, and then 5 hr later we start to see appearance of the fluorescence in the locating caspase-2 activation. This increases in intensity over time until it finally reaches a threshold. Within the same experiments we also treated cells at a sublethal dose of heat shock for 1 hr at 42°C and these cells didn't undergo apoptosis, and they still were able to divide and we didn't see any caspase-2 activation. So to show that this technique is specific for caspase-2, we disrupted the ability of caspase-2 to bind to its activation platform by disrupting its ability to bind to RAIDD.
So in 1998, it was published that there are two amino acids on the surface of the caspase-2 CARD, E87 and D83, that appear to be responsible for the binding to RAIDD. A mutation of these was reported to disrupt the binding between these two proteins. So we introduced both of these mutations into our caspase-2 CARD BiFC pair, and we first did co-immunoprecipitation experiments to verify that the mutation did in fact disrupt the binding. So here we have expressed the wild-type CARD which is HA tag, with FLAG tag RAIDD, and we immunoprecipitated the FLAG RAIDD and blotted four and caspase-2 CARD, and we see robust binding between caspase-2 CARD and RAIDD. When we expressed the mutants we were still able to detect some binding, although it was much lower. So the mutation of these two residues doesn't completely abrogate the binding, it just merely disrupts it. Consistent with the weaker binding, we saw much lower efficiency of caspase-2 BiFC in response to heat shock when we expressed the mutant caspase-2 CARD, compared to the wild-type caspase-2 CARD.
To further confirm this, we used RAIDD knockout cells, so we used RAIDD wild-type and knockout litter-matched mouse embryonic cytoblasts. In the wild-type cells, as expected, after heat shock we saw induction of caspase-2 fluorescence complementation. In the knockouts we didn't see any fluorescence complementation induced by heat shock, so this shows that RAIDD was required for caspase-2 activation. To confirm that this was dependent on RAIDD, we re-expressed RAIDD in these cells and were able to restore the ability of the cells to induce caspase-2 BiFC in response to heat shock. So these results told us two things, that RAIDD is required for caspase-2 BiFC, so we can conclude that the BiFC technique is specific for caspase-2, and recruitment of caspase-2 to its activation platform after heat shock is dependent on RAIDD.
Our next question was, where is caspase-2 activated? So here's another movie, which is just a little bit more detailed than the last one. What you will see is the green spots representing caspase-2 activation will start to form in the peripheries of the cell, and move in to a region that is adjacent to the nucleus. They accumulate all in this region that is beside the nucleus. The fact that caspase-2 wasn't in the nucleus was surprising to us, because caspase-2 has a nuclear localization sequence and has been reported to be expressed in the nucleus. So we went back and we looked at our caspase-2 CARD, BiFC constructs and we realized that we hadn't included the NLS in there. So we went back and we made caspase-2 pro-version, and we also made a version of caspase-2 full-length, and we made this catalytically inactive, and we changed it from an active cysteine to an alanine so that it wouldn't kill the cells when we overexpressed it.
We made 3D reconstructions of the cell so we could determine precisely where caspase-2 was being activated. So here is the 3D reconstructions and this is on untreated cells expressing the caspase-2 full-length pair, there's no fluorescence, no green fluorescence. The red is the mitochondria and the blue is the nucleus. Then when we heat shocked the cells we see appearance of green representing caspase-2 activation, and this is exclusively in the cytosol and it's also not associated with the mitochondria. When we expressed the caspase-2 pro pair, we see the same distribution exclusively in the science model and not nuclear, and the caspase-2 CARD pair gave us the same distribution as well. So these results suggest that the caspase-2 was activated in the nucleus, and we also saw similar results with other treatments such as DNA damage and cytoskeletal disruption.
The next question was, when in the apoptotic cascade is caspase-2 activated? So caspase-2 has been shown to be cleaved upstream of the mitochondria, and downstream of caspase-3. To determine which of these represented activation, we overexpressed Bcl-xL which blocks the downstream events. So HeLa cells that are overexpressed Bcl-xL are potently protected from heat shock induced cell death. But, in contrast, when we looked at caspase-2 BiFC in response to heat shock, there was no difference in caspase-2 activation in HeLa cells versus HeLa cells overexpressing Bcl-xL. So this indicated that caspase-2 is activated in the absence of apoptosis.
To look more directly at the relationship between caspase-2 and mitochondria permeabilization, we used a marker for MOMP and this is Omi-mCherry. So Omi is a protein which like cytochrome c lives in the intermembrane space of the mitochondria, and gets released from the mitochondria at the same time as cytochrome c. This is here it's fused to a red fluorescent protein called mCherry, so we see it in red and at the beginning of the movie on the mitochondria, and we heat shocked the cells and, as before, we see induction of caspase-2 activation. This occurs long before release of Omi, so in the upper cell you'll soon see the Omi go from mitochondrial to diffuse, and the lower cell it doesn't release its Omi in the time of this movie.
We can, again, express this graphically and these are three individual cells, so in the red as you see the graph goes from high values, this is punctate or when it's in the mitochondria, to low where it becomes a more diffuse pattern. We can pinpoint exactly when Omi release occurs by this dropdown in the graph. In both of these cells, caspase-2 activation starts well before Omi release occurs, and in the third cell, even though we get robust caspase-2 activation, there's no release of Omi. So these experiments together strongly suggest that caspase-2 is activated upstream of mitochondria permeabilization.
Finally, we wanted to use this technique to determine how caspase-2 is regulated, and we looked first for the heat shock pathway. So heat shock as well as inducing apoptosis, also engages survival factors such by activating the transcription factor HSF-1, standing for Heat Shock Factor 1. HSF-1 leads to the upregulation of the number of heat shock proteins, so we wanted to know if these heat shock proteins could inhibit caspase-2 activation. So, as I said, HSF-1 is a survival factor and HSF-1 knockout cells are much more sensitive to heat shock induced apoptosis in the wild-type cells.
We did similar experiments, except this time looking at caspase-2 BiFC, so after 43° heat shock, which is a kind of a low temperature, we didn't see any activation of caspase-2 in the HSF-1 wild-type cells, but we saw an increase in caspase-2 activation in the knockouts. So the reasons that this might be HSF-1 is upregulating something that's inhibiting caspase-2 activation and we had a good inhibitor for Hsp90, which is 17 DMAG. So when we added that in the HSF-1 repeat cells we were able to now activate caspase-2 in response to heat shock, suggesting that Hsp90 is inhibiting caspase-2 activation.
To confirm that this was Hsp90, and in particular more specifically Hsp90 alpha, which is the inducible form of Hsp90, we used siRNA to knockdown Hsp90. So here is the siRNA, which gave pretty efficient knockdown and under controlled conditions where we had either no RNA, or controlled siRNA we didn't see any caspase-2 activation. But when we knocked down Hsp90 the cells were now able to activate caspase-2 in response to heat shock.
So to summarize this, HSF-1 blocks caspase-2 activation and seems to do so by upregulating the inducible form of Hsp90, Hsp90 alpha, which blocks caspase-2 activation. We can overcome this inhibition by blocking Hsp90 activity either with the chemical inhibitor 17-DMAG, or Hsp90 alpha siRNA restoring caspase-2 activation.
So in the last couple of slides I just want to talk a bit about some of the - how this work has allowed us to look at non-apoptotic roles of caspase-2 and, in particular, we were interested in the fact that caspase-2 can act as a tumor suppressor. So recently we reported that caspase-2 appears to be a tumor suppressor in a mammary tumor model. So we used the MMTV/c-neu mouse model where the oncogene, c-neu, is expressed off the mammary-specific MMTV promoter. These mice, after about a year, 60% of the mice had come to tumors, but when we knocked out caspase-2 we saw significantly accelerated tumor onset and growth. So caspase-2 acts as a tumor suppressor in the mammary tumor model, and it had previously been reported that it acts as a tumor suppressor in a mouse model with lymphoma.
To see how caspase-2 is suppressing these tumors, we looked at the tumor sections and we classified these into four groups going from least abnormal to most abnormal. So the first was mild karyomegaly where the nuclei were slightly enlarged. The second was rarely multinucleated, and these two categories mostly showed up in the caspase-2 wild-type tumors. The next group was occasionally multinucleated with karyomegaly, and finally the most abnormal group had abnormal multinucleated cells with karyomegaly and bizarre mitotic figures. We only ever saw these in the caspase-2 knockout tumors, and many of these abnormalities, including bizarre mitotic figures, which you can see annotated by the blue arrows, are consistent with cell cycle defects. So this led us to think that maybe caspase-2 was inhibiting cell cycle in some way in these tumors.
So to confirm this we looked up the mitotic index in the tumors, and in the caspase-2 knockouts there was significantly more cells undergoing mitosis than in the wild-types. So caspase-2 may suppress tumors not just by inducing apoptosis, but also by regulating cell cycle. We hypothesized that this regulation of cell cycle may be to prevent accumulation of DNA damage in tumor cells, and this would otherwise lead to genomic instability.
So to test for this, we used an assay called the PALA assay, and PALA is the pyrimidine synthase inhibitor which leads to arrest in G1. Under normal circumstances this results in death of the cells, but if there is genomic instability, the cells will amplify certain parts of their DNA and they're more likely to amplify a gene called CAD; and CAD encodes for an enzyme which is essential for UMP synthesis, and is the only way of overcoming the toxic effects of PALA, so the cells grow out.
So we tested this on our caspase-2 wild-type and knockout E1A transformed mouse embryonic fibroblasts, and the results were quite striking. So we treated the cells with either three times or five times their LD50 doses of PALA, and only in the caspase-2 knockouts were the cells able to grow out. We collected the cells and looked for amplification of CAD by real-time PCR, and in the caspase-2 knockouts there was an increase in the amount of CAD as present, indicating amplification of CAD. So these results suggest that caspase-2 protects against genomic instability.
To determine the mechanism of this, we first looked at the p53 response, because p53 is a known sentinel of genomic stability. So we treated caspase-2 wild-type and caspase-2 knockout cells with PALA, and we looked at a number of p53 target genes. Among these were Mdm2 and p21, so while these were robustly induced in caspase-2 wild-type cells and caspase-2 was knocked out, the expression of these genes was greatly impaired. So this indicates that caspase-2 associated protection from genomic instability, may be p53 dependent.
So to conclude from this talk and to summarize that we have discussed direct imaging of caspase activation and activity in real-time, is a highly-informative way to investigate functions of caspases in apoptosis and non-apoptotic events. It allows caspase activation and consequence of that activation to be interrogated in the same cell. We specifically discussed caspase BiFC, which can be used to dissect the activation platform requirements for initiating caspase activation. To determine the kinetics of initiating caspase activation, as well as the subcellular localization and organization of activation platforms. This can be very useful to investigating upstream regulatory factors of initiator caspase activation. We use this specifically to interrogate the caspase-2 pathway, which appears to act as a tumor suppressor in a mammary tumor model, and function to ensure correct cell cycle progression to prevent accumulation of genomic instability.
I'll just finish by thanking the people in my lab: Melissa Parsons, Mason Sanders and Sara Fassio, and also from St Jude's Children's Research Hospital where many of these projects originated. A lot of this work was done in the Green's Lab, and a number of people contributed to the success of those projects. Thank you for your attention, I will be back shortly to answer your questions. I'm going to handover to David, who has a few slides about Abcam.
DB: Thank you, Lisa, for a really great talk. Hello, I would like to take this opportunity to tell you quickly about some resources and products that Abcam has available for your cancer research. Our products can be reviewed by our customers, we call these reviews AbReviews. AbReviews are unbiased where we publish all positive and negative reviews, and these are also quick and easy to complete.
Abcam has a dedicated multilingual scientific support to help if you have any problems or questions about our products. All of our products are covered by our AbPromise where we guarantee that a product will work in an application of species has been listed on our datasheet. We also offer testing discounts when a species or application are not listed on our reagents datasheet.
Abcam offers the largest range of rabbit monoclonal antibodies, and these combine the benefits of monoclonal specificity with the robust rabbit immune system. Since these are raised in rabbit they show no cross-reactivity when using on mouse tissue, so why not try our RabMAb from Abcam today.
We also have a broad range of secondary antibodies to complement our primary antibodies, including our new range of Alexa Fluor® conjugated secondaries. These are available pre-adsorbed to different species and in a range of Alexa Fluor® conjugates.
We also offer self-staining reagents and our CytoPainter range or reagents is ideal for multicolor staining. It allows you to visualize subcellular components, such as the mitochondria or lysosomes, and these can be used in fixed as well as live cells.
We have a number of western blotting antibody cocktails, including our apoptosis cocktail, and these reagents allow you to detect multiple proteins in blot. We also have apoptosis detection kits that monitor the flipping of phosphatidylserine from the interface of the plasma membrane to the cell surface after apoptosis is initiated.
We launched a range of ELISA kits last year, our SimpleStep ELISA kits. The time taken to develop our SimpleStep ELISA kits is reduced when compared to the traditional ELISAs, this is because our new kits require only one washing step. This reduction in washes also improves the sensitivity and specificity of these kits.
Abcam offers a wide range of pharmacological tools, and these include over 4,000 enzyme receptor and ion channel inhibitors and activators. Our range of pharmacological tools include some of the compounds Lisa spoke about today, including 17-DMAG, actinomycine d and various caspase substrates and inhibitors.
We are currently pulling together our different cancer research resources onto one page at abcam.com/cancer. We've brought together products, articles, pathways, as well as relevant webinars and events. This is a work-in-progress and we're aiming to continually improve this page.
I would like to highlight our upcoming Crossing Boundaries: Linking Metabolism to Epigenetics meeting, taking place in Boston, Massachusetts in May. If you'd like to find out more information about this meeting please visit our website at abcam.com/CrossingBoundaries.
We are currently running a promotion on our primary antibodies where you can buy two primaries, and receive one rabbit monoclonal antibody of your choice for free. This promotion lasts until Friday, 14th March and you can redeem this using the following code. This will be through our website or just go to abcam.com/promotions. You will receive this in an email after the webinar and details are on this special offer, as well as a PDF file of Lisa's slides. I'll now pass you back over to Lisa who is ready to answer your questions we received during the webinar. Thanks for your attention.
LB-H: So thank you, David, and we've had a few good questions, which I'll try to answer. The first is from Patricia Fernandez, and she asked: As far as I understand, the IFC is exclusive for caspase-2, is it not possible to develop for caspase-3 or caspase-1? This is a really good question. We have only been successful with caspase-2 so far, we are currently developing it in the lab for other caspases. But it's important to note that this will only work for initiator caspases that are activated by dimerization, so if you're talking about caspase-3, remember it's already in the cell as a preformed dimer. So because it doesn't get activated by dimerization, the IFC technique wouldn't really work for this. So, in that case, using the FRET-based techniques would be a much more beneficial approach.
Another question is from Alex Robson, and he asks: Using the stressors mentioned, have you compared the caspase-2 BiFC localization to endogenous caspase-2 localization using immunofluorescence, and do we see a similar signal? Yes, we have and, as I mentioned, if you do immunofluorescence for endogenous caspase-2, you see it coming up in the nucleus, the cytosol and the Golgi, so it's everywhere. So our results suggest that the active portion of caspase-2 doesn't appear to be in the nucleus, even though caspase-2 exists there. There could be a number of reasons for this, it could mean that caspase-2 has functions in the nucleus that have nothing to do with its activation, or it could mean that caspase-2 goes to a nucleus after it's activated, and cleaves substrates there. So these are really great open questions, and we have not so far seen the same punctate signal of endogenous caspase-2, and I think we haven't really looked at it very extensively. So I think it's because what we're looking at with the IFC is just the portion of caspase-2 that has been activated, whereas when you look at endogenous caspase-2 this is overcome by the rest of the caspase-2 that is there. So far we haven't seen it, but we are still working on those kind of experiments.
Alex also asks if we've compared caspase-2 BiFC to caspase-8 under the same conditions? This is similar to what Patricia asked us; and, yes, we tried to do that, but the caspase-8 construct that we were using used the same prodomain strategy. The prodomain of caspase-8, which includes two death effector domains, when we expressed this it had a really high background BiFC, and it was also highly toxic to the cells.So we were unable to do any further analysis with this, so each caspase that you look at has to be really detailed, really explored on their own and not in comparison to other ones. So we think maybe by using different truncations of caspase-8 we may be able to solve that some day, but we haven't solved it just yet. I think that is all we have time for, so I'm going to hand you back to Lucy, and thank you very much for your attention.
LP: Thank you, Lisa and David for your presentations. As Lisa mentioned, unfortunately, due to time restrictions we're not able to answer all the questions received. For those whose questions were not answered we will be in contact with you shortly with a response. Just to let you know when you log-off from this webinar, you will be redirected to a webpage where more information can be found about the three for two offer. Also, a PDF of today's presentations will be emailed to you shortly. If you do have any further questions about what we have discussed today or have any technical enquiry, please don't hesitate to contact our scientific support team who will be very happy to help you. They can be emailed at firstname.lastname@example.org. We hope you have found this webinar informative and useful to your work, and we look forward to welcoming you to another webinar in the future. Thank you again for attending, and good luck with your research!