Detection of endoplasmic reticulum stress and the unfolded protein response in naturally-occurring endocrinopathic equine laminitis.

BACKGROUND: Laminitis is often associated with endocrinopathies that cause hyperinsulinemia and is also induced experimentally by hyperinsulinemia, suggesting that insulin initiates laminitis pathogenesis. Hyperinsulinemia is expected to activate pro-growth and anabolic signaling pathways. We hypothesize that chronic over-stimulation of these pathways in lamellar tissue results in endoplasmic reticulum stress, contributing to tissue pathology, as it does in human metabolic diseases. We tested this hypothesis by asking whether lamellar tissue from horses with naturally-occurring endocrinopathic laminitis showed expression of protein markers of endoplasmic reticulum stress. RESULTS: Three markers of endoplasmic reticulum stress, spliced XBP1, Grp78/BiP and Grp94, were upregulated 2.5-9.5 fold in lamellar tissues of moderately to severely laminitic front limbs (n = 12) compared to levels in controls (n = 6-7) measured by immunoblotting and densitometry. Comparing expression levels between laminitic front limbs and less affected hind limbs from the same horses (paired samples from 7 to 8 individual horses) demonstrated significantly higher expression for both spliced XBP1 and Grp78/BiP in the laminitic front limbs, and a similar trend for Grp94. Expression levels of the 3 markers were minimal in all samples of the control (n = 6-7) or hind limb groups (n = 7-8). Immunofluorescent localizations were used to identify cell types expressing high levels of Grp78/BiP, as an indicator of endoplasmic reticulum stress. Grp78/BiP expression was highly elevated in suprabasal epidermal keratinocytes and only observed in laminitic front limbs (10/12 laminitic samples, compared to 0/7 in sections from the hind limbs and 0/5 of controls). CONCLUSIONS: These data demonstrate that the endoplasmic reticulum stress pathway is active in naturally occurring cases of laminitis and is most active within a subset of epidermal keratinocytes. These data provide the rationale for further study of endoplasmic reticulum stress in experimental models of laminitis and the links between laminitis and human diseases sharing activation of this stress pathway. Pharmacological options to manipulate the endoplasmic reticulum stress pathway under investigation for human disease could be applicable to laminitis treatment and prevention should this pathway prove to be a driver of disease progression.

Tubulin delivery: polymerization chaperones for microtubule assembly?

A new paper by Slep and Vale in a recent issue of Molecular Cell provides structural clues as to how three different +TIP proteins interact with tubulin and suggests that +TIPs deliver oligomers of tubulin dimers to growing microtubule ends.

The oncoprotein 18/stathmin family of microtubule destabilizers.

The past several years have seen major advances in our understanding of the mechanisms of microtubule destabilization by oncoprotein18/stathmin (Op18/stathmin) and related proteins. New structural information has clearly shown how members of the Op18/stathmin protein family bind tubulin dimers and suggests models for how these proteins stimulate catastrophe, the transition from microtubule growth to shortening. Regulation of Op18/stathmin by phosphorylation continues to capture much attention. Studies suggest that phosphorylation occurs in a localized fashion, resulting in decreased microtubule destabilizing activity near chromatin or microtubule polymer. A spatial gradient of inactive Op18/stathmin associated with chromatin or microtubules could contribute significantly to mitotic spindle assembly.

Continuous digital hypothermia reduces expression of keratin 17 and 1L-17A inflammatory pathway mediators in equine laminitis induced by hyperinsulinemia.

The euglycemic hyperinsulinemic clamp model (EHC) of equine endocrinopathic laminitis induces rapid loss of lamellar tissue integrity, disrupts keratinocyte functions, and induces inflammation similar to natural disease. Continuous digital hypothermia (CDH) blocks tissue damage in this experimental model, allowing identification of specific genes or molecular pathways contributing to disease initiation or early progression. Archived lamellar tissues (8 horses, 48 h EHC treatment, including CDH-treated front limbs) were used to measure relative expression levels of genes encoding keratin 17 (KRT17), a stress-induced intermediate filament protein, and genes upregulated downstream of keratin 17 and/or interleukin 17A (IL-17A), as mediators of inflammation. Compared to front or hind limbs at ambient temperature, CDH resulted in significantly lower expression of KRT17, CCL2, CxCL8, PTGS2 (encoding COX2), IL6, TNFα, S100A8 and MMP1. By immunofluorescence, COX2 was robustly expressed in lamellar keratinocytes from ambient limbs, but not in CDH-treated limbs. Genes not significantly reduced by CDH were IL17A, DEFB4B, S100A9 and MMP9. Overall, 8 of 12 genes were expressed at lower levels in the CDH-treated limb. These 8 genes are expressed by wounded or stress-activated keratinocytes in human disease or mouse models, highlighting the role of keratinocytes in equine laminitis.

TOGp regulates microtubule assembly and density during mitosis and contributes to chromosome directional instability.

TOGp, a member of the XMAP215 MAP family, is required for bipolar mitotic spindle assembly. To understand how TOGp contributes to spindle assembly, we examined microtubule dynamics after depleting TOGp by siRNA. Fluorescence recovery after photobleaching of GFP-tubulin demonstrated that spindle microtubule turnover is slowed two-fold in the absence of TOGp. Consistent with photobleaching results, microtubule regrowth after washout of the microtubule depolymerizing drug nocodazole was slower at the centrosomes and in the vicinity of mitotic chromatin in cells depleted of TOGp. The slower microtubule turnover is likely due to either nucleation or the transitions of dynamic instability because TOGp depletion did not effect the rate of plus end growth, measured by tracking EB1-GFP at microtubule ends. In contrast, microtubule regrowth after nocodazole washout was unaffected by prior depletion of TACC3, a centrosomal protein that interacts with TOGp. Kinetochore fibers in both untreated and TOGp-depleted cells were stable to incubation at 4 degrees C or lysis in buffer containing calcium indicating that stable kinetochore-microtubule attachments are formed in the absence of TOGp. Depletion of TOGp, but not TACC3, reduced kinetochore oscillations during prometaphase/metaphase. Defects in oscillations are not due simply to multipolarity or loss of centrosome focus in the TOGp-depleted cells, since kinetochore oscillations appear normal in cells treated with the proteosome inhibitor MG132, which also results in multipolar spindles and centrosome fragmentation. We hypothesize that TOGp is required for chromosome motility as a downstream consequence of reduced microtubule dynamics and/or density. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Interleukin-17A pathway target genes are upregulated in Equus caballus supporting limb laminitis.

Supporting Limb Laminitis (SLL) is a painful and crippling secondary complication of orthopedic injuries and infections in horses, often resulting in euthanasia. SLL causes structural alterations and inflammation of the interdigitating layers of specialized epidermal and dermal tissues, the lamellae, which suspend the equine distal phalanx from the hoof capsule. Activation of the interleukin-17A (IL-17A)-dependent inflammatory pathway is an epidermal stress response that contributes to physiologic cutaneous wound healing as well as pathological skin conditions. As a first test of the hypothesis that hoof lamellae of horses diagnosed with SLL also respond to stress by activating the IL-17A pathway, the expression of IL-17A, IL-17 receptor subunit A and 11 IL-17A effector genes was measured by RT-PCR or qPCR. Lamellar tissue was isolated from Thoroughbreds euthanized due to naturally occurring SLL and in age and breed matched non-laminitic controls. By RT-PCR, the IL-17 Receptor A subunit was expressed in both non-laminitic and laminitic tissues, while IL-17A was primarily detectable in laminitic tissues. IL-17A target gene expression was undetectable in non-laminitic samples with the exception of weak detection of DEFB4B, S100A9 and PTSG2. In contrast, all target genes examined, except CCL20, were expressed by some or all laminitic samples. By qPCR, severe acute (n = 7) SLL expressed ~15-100 fold higher levels of DEFB4B and S100A9 genes compared to non-laminitic controls (n = 8). DEFB4B was also upregulated in developmental/subclinical (n = 8) and moderate acute (n = 7) by ~ 5-fold, and in severe chronic (n = 5) by ~15-200 fold. In situ hybridization (DEFB4) and immunofluorescence (calprotectin, a dimer of S100A9/S100A8 proteins) demonstrated expression in keratinocytes, primarily in suprabasal cell layers, from SLL samples. These data demonstrate upregulation of a cohort of IL-17A target genes in SLL and support the hypothesis that similarities in the response to stresses and damage exist between equine and human epidermal tissues.

Gene expression profiles in mouse embryo fibroblasts lacking stathmin, a microtubule regulatory protein, reveal changes in the expression of genes contributing to cell motility.

BACKGROUND: Stathmin (STMN1) protein functions to regulate assembly of the microtubule cytoskeleton by destabilizing microtubule polymers. Stathmin over-expression has been correlated with cancer stage progression, while stathmin depletion leads to death of some cancer cell lines in culture. In contrast, stathmin-null mice are viable with minor axonopathies and loss of innate fear response. Several stathmin binding partners, in addition to tubulin, have been shown to affect cell motility in culture. To expand our understanding of stathmin function in normal cells, we compared gene expression profiles, measured by microarray and qRT-PCR, of mouse embryo fibroblasts isolated from STMN1+/+ and STMN1-/- mice to determine the transcriptome level changes present in the genetic knock-out of stathmin. RESULTS: Microarray analysis of STMN1 loss at a fold change threshold of > or = 2.0 revealed expression changes for 437 genes, of which 269 were up-regulated and 168 were down-regulated. Microarray data and qRT-PCR analysis of mRNA expression demonstrated changes in the message levels for STMN4, encoding RB3, a protein related to stathmin, and in alterations to many tubulin isotype mRNAs. KEGG Pathway analysis of the microarray data indicated changes to cell motility-related genes, and qRT-PCR plates specific for focal adhesion and ECM proteins generally confirmed the microarray data. Several microtubule assembly regulators and motors were also differentially regulated in STMN1-/- cells, but these changes should not compensate for loss of stathmin. CONCLUSION: Approximately 50% of genes up or down regulated (at a fold change of > or = 2) in STMN1-/- mouse embryo fibroblasts function broadly in cell adhesion and motility. These results support models indicating a role for stathmin in regulating cell locomotion, but also suggest that this functional activity may involve changes to the cohort of proteins expressed in the cell, rather than as a direct consequence of stathmin-dependent regulation of the microtubule cytoskeleton.

Mitosis: riding the protofilament curl.

More than 50 years ago, microtubule depolymerization was proposed as the force responsible for chromosome movement. New studies measure the force produced by depolymerization and show that protein ring complexes can couple depolymerization to movement. These results have implications for anaphase chromosome motility and mitotic evolution.

Infection with replication-deficient adenovirus induces changes in the dynamic instability of host cell microtubules.

Adenovirus translocation to the nucleus occurs through a well characterized minus end-directed transport along microtubules. Here, we show that the adenovirus infection process has a significant impact on the stability and dynamic behavior of host cell microtubules. Adenovirus-infected cells had elevated levels of acetylated and detyrosinated microtubules compared with uninfected cells. The accumulation of modified microtubules within adenovirus-infected cells required active RhoA. Adenovirus-induced changes in microtubule dynamics were characterized at the centrosome and at the cell periphery in living cells. Adenovirus infection resulted in a transient enhancement of centrosomal microtubule nucleation frequency. At the periphery of adenovirus-infected cells, the dynamic instability of microtubules plus ends shifted toward net growth, compared with the nearly balanced growth and shortening observed in uninfected cells. In infected cells, microtubules spent more time in growth, less time in shortening, and underwent catastrophes less frequently compared with those in uninfected cells. Drug-induced inhibition of Rac1 prevented most of these virus-induced shifts in microtubule dynamic instability. These results demonstrate that adenovirus infection induces a significant stabilizing effect on host cell microtubule dynamics, which involve, but are not limited to, the activation of the RhoGTPases RhoA and Rac1.

Reorganization of paclitaxel-stabilized microtubule arrays at mitotic entry: roles of depolymerizing kinesins and severing proteins.

Paclitaxel is a widely used anti-cancer treatment that disrupts cell cycle progression by blocking cells in mitosis. The block at mitosis, with spindles assembled from short microtubules, is surprising given paclitaxel's microtubule stabilizing activity and the need to depolymerize long interphase microtubules prior to spindle formation. Cells must antagonize paclitaxel's microtubule stabilizing activity during a brief window of time at the transition from interphase to mitosis, allowing microtubule reorganization into a mitotic spindle, although the mechanism underlying microtubule depolymerization in the presence of paclitaxel has not been examined. Here we test the hypothesis that microtubule severing and/or depolymerizing proteins active at mitotic entry are necessary to clear the interphase array in paclitaxel-treated cells and allow subsequent formation of mitotic spindles formed of short microtubules. A549 and LLC-PK1 cells treated with 30nM paclitaxel approximately 4 h prior to mitotic entry successfully progress through the G2/M transition by clearing the interphase microtubule array from the cell interior outward to the cell periphery, a spatial pattern of reorganization that differs from that of cells possessing dynamic microtubules. Depletion of kinesin-8s, KIF18A and/or KIF18B obstructed interphase microtubule clearing at mitotic entry in paclitaxel-treated cells, with KIF18B making the larger contribution. Of the severing proteins, depletion of spastin, but not katanin, reduced microtubule loss as cells entered mitosis in the presence of paclitaxel. These results support a model in which KIF18A, KIF18B, and spastin promote interphase microtubule array disassembly at mitotic entry and can overcome paclitaxel-induced microtubule stability specifically at the G2/M transition.

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus.

The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are lamellar tissue-specific and could serve as differentiation- and disease-specific markers. Our objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three monoclonal antibodies generated against N- and C-terminal K124 peptides detected a band of the appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-K124 monoclonal antibodies.

Cytoskeleton: microtubules born on the run.

The organization of microtubules into large arrays determines cell morphology and structure. Recent work in the fission yeast describes a novel mechanism for microtubule self-organization in the absence of centrosomes; this mechanism may function in a variety of cell types found in diverse organisms.

Cellular entry and nuclear targeting by a highly anionic molecular umbrella.

A fluorescently labeled, persulfated molecular umbrella ( 1) has been synthesized from cholic acid, lysine, spermine, and Coumarin 343 and found capable of entering live HeLa cells. The distributions of 1 throughout the cytoplasm and the nucleus were diffuse and punctate, respectively. This finding, together with its ability to cross liposomal membranes by passive diffusion, suggests that passive diffusion plays a significant role in the ability of 1 to enter cells. The fact that 1 is concentrated at the nucleus raises the possibility that molecular umbrellas of this type could be used for the nuclear targeting of drugs.

Monte Carlo simulations of microtubule arrays: The critical roles of rescue transitions, the cell boundary, and tubulin concentration in shaping microtubule distributions.

Microtubules are dynamic polymers required for a number of processes, including chromosome movement in mitosis. While regulators of microtubule dynamics have been well characterized, we lack a convenient way to predict how the measured dynamic parameters shape the entire microtubule system within a cell, or how the system responds when specific parameters change in response to internal or external signals. Here we describe a Monte Carlo model to simulate an array of dynamic microtubules from parameters including the cell radius, total tubulin concentration, microtubule nucleation rate from the centrosome, and plus end dynamic instability. The algorithm also allows dynamic instability or position of the cell edge to vary during the simulation. Outputs from simulations include free tubulin concentration, average microtubule lengths, length distributions, and individual length changes over time. Using this platform and reported parameters measured in interphase LLCPK1 epithelial cells, we predict that sequestering ~ 15-20% of total tubulin results in fewer microtubules, but promotes dynamic instability of those remaining. Simulations also predict that lowering nucleation rate will increase the stability and average length of the remaining microtubules. Allowing the position of the cell's edge to vary over time changed the average length but not the number of microtubules and generated length distributions consistent with experimental measurements. Simulating the switch from interphase to prophase demonstrated that decreased rescue frequency at prophase is the critical factor needed to rapidly clear the cell of interphase microtubules prior to mitotic spindle assembly. Finally, consistent with several previous simulations, our results demonstrate that microtubule nucleation and dynamic instability in a confined space determines the partitioning of tubulin between monomer and polymer pools. The model and simulations will be useful for predicting changes to the entire microtubule array after modification to one or more parameters, including predicting the effects of tubulin-targeted chemotherapies.

Cell division: eg'ing on microtubule flux.

Mitotic microtubules are moved toward spindle poles in a process known as flux. Several proteins responsible for flux have recently been identified, significantly advancing our understanding of chromosome movements in mitosis.

TOGp, the human homolog of XMAP215/Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly.

The XMAP215/Dis1 MAP family is thought to regulate microtubule plus-end assembly in part by antagonizing the catastrophe-promoting function of kin I kinesins, yet XMAP215/Dis1 proteins localize to centrosomes. We probed the mitotic function of TOGp (human homolog of XMAP215/Dis1) using siRNA. Cells lacking TOGp assembled multipolar spindles, confirming results of Gergely et al. (2003. Genes Dev. 17, 336-341). Eg5 motor activity was necessary to maintain the multipolar morphology. Depletion of TOGp decreased microtubule length and density in the spindle by approximately 20%. Depletion of MCAK, a kin I kinesin, increased MT lengths and density by approximately 20%, but did not disrupt spindle morphology. Mitotic cells lacking both TOGp and MCAK formed bipolar and monopolar spindles, indicating that TOGp and MCAK contribute to spindle bipolarity, without major effects on MT stability. TOGp localized to centrosomes in the absence of MTs and depletion of TOGp resulted in centrosome fragmentation. TOGp depletion also disrupted MT minus-end focus at the spindle poles, detected by localizations of NuMA and the p150 component of dynactin. The major functions of TOGp during mitosis are to focus MT minus ends at spindle poles, maintain centrosome integrity, and contribute to spindle bipolarity.

Organization and dynamics of growing microtubule plus ends during early mitosis.

A stable cell line expressing EB1-green fluorescent protein was used to image growing microtubule plus ends at the G(2)/M transition. By late prophase growing ends no longer extend to the cell periphery and were not uniformly distributed around each centrosome. Growing ends were much more abundant in the area surrounding the nuclear envelope, and microtubules growing around the nucleus were 1.5 fold longer than those growing in the opposite direction. The growth of longer ends toward the nucleus did not result from a localized faster growth rate, because this rate was approximately 11 microm/min in all directions from the centrosome. Rather, microtubule ends growing toward the nucleus seemed stabilized by dynein/dynactin associated with the nuclear envelope. Injection of p50 into late prophase cells removed dynein from the nuclear envelope, reduced the density of growing ends near the nuclear envelope and resulted in a uniform distribution of growing ends from each centrosome. We suggest that the cell cycle-dependent binding of dynein/dynactin to the nuclear envelope locally stabilizes growing microtubules. Both dynein and microtubules would then be in a position to participate in nuclear envelope breakdown, as described in recent studies.

Human EFO1p exhibits acetyltransferase activity and is a unique combination of linker histone and Ctf7p/Eco1p chromatid cohesion establishment domains.

Proper segregation of chromosomes during mitosis requires that the products of chromosome replication are paired together-termed sister chromatid cohesion. In budding yeast, Ctf7p/Eco1p is an essential protein that establishes cohesion between sister chromatids during S phase. In fission yeast, Eso1p also functions in cohesion establishment, but is comprised of a Ctf7p/Eco1p domain fused to a Rad30p domain (a DNA polymerase) both of which are independently expressed in budding yeast. In this report, we identify and characterize the first candidate human ortholog of Ctf7p/Eco1p, which we term hEFO1p (human Establishment Factor Ortholog). As in fission yeast Eso1p, the hEFO1p open reading frame extends well upstream of the C-terminal Ctf7p/Eco1p domain. However, this N-terminal extension in hEFO1p is unlike Rad30p, but instead exhibits significant homology to linker histone proteins. Thus, hEFO1p is a unique fusion of linker histone and cohesion establishment domains. hEFO1p is widely expressed among the tissues tested. Consistent with a role in chromosome segregation, hEFO1p localizes exclusively to the nucleus when expressed in HeLa tissue culture cells. Moreover, biochemical analyses reveal that hEFO1p exhibits acetyltransferase activity. These findings document the first characterization of a novel human acetyltransferase, hEFO1p, that is comprised of both linker histone and Ctf7p/Eco1p domains.

The chemical biology of Cu(II) complexes with imidazole or thiazole containing ligands: Synthesis, crystal structures and comparative biological activity.

The synthesis and characterization of two copper(II) complexes containing 2-(2-pyridyl)benzimidazole (PyBIm) are reported with the biological activity of these two complexes and a third Cu(II) complex containing 2-(2-pyridyl)benzothiazole (PyBTh). Complex 1, [Cu(PyBIm)(NO3)(H2O)](NO3), is a four coordinate, distorted square planar species with one ligand (N,N), nitrate and water bound to Cu(II). The [Cu(PyBIm)3](BF4)2 complex (2) has distorted octahedral geometry with a 3:1 Py(BIm) ligand to metal ratio. The distorted trigonal bi-pyramidal geometry of compound 3, [Cu(PyBTh)2(H2O)](BF4)2, is comprised of two PyBTh ligands and one water. Biological activity of 1-3 has been assessed by analyzing DNA interaction, nuclease ability, cytotoxic activity and antibacterial properties. Complex 3 exhibits potent concentration dependent SC-DNA cleavage forming single- and double-nicked DNA in contrast to the weak activity of complexes 1 and 2. Mechanistic studies indicate that all complexes utilize an oxidative mechanism however 1 and 2 employ O2(-) as the principal reactive oxygen species while the highly active 3 utilizes (1)O2. The interaction between 1-3 and DNA was investigated using fluorescence emission spectroscopy and revealed all complexes strongly intercalate DNA with Kapp values of 2.65 × 10(6), 1.85 × 10(6) and 2.72 × 10(6)M(-1), respectively. Cytotoxic effects of 1-3 were examined using HeLa and K562 cells and show cell death in the micromolar range with the activity of 1 ≈ 2 and were slightly higher than 3. Similar reactivity was observed in the antibacterial studies with E. coli and S. aureus. A detailed comparative analysis of the three complexes is presented.

A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells.

Stathmin/Oncoprotein 18, a microtubule destabilizing protein, is required for survival of p53-deficient cells. Stathmin-depleted cells are slower to enter mitosis, but whether delayed mitotic entry triggers cell death or whether stathmin has a separate pro-survival function was unknown. To test these possibilities, we abrogated the cell cycle delay by inhibiting Wee1 in synchronized, stathmin-depleted cells and found that apoptosis was reduced to control levels. Synchronized cells treated with a 4 hour pulse of inhibitors to CDK1 or both Aurora A and PLK1 delayed mitotic entry and apoptosis was triggered only in p53-deficient cells. We did not detect mitotic defects downstream of the delayed mitotic entry, indicating that cell death is activated by a mechanism distinct from those activated by prolonged mitotic arrest. Cell death is triggered by initiator caspase 8, based on its cleavage to the active form and by rescue of viability after caspase 8 depletion or treatment with a caspase 8 inhibitor. In contrast, initiator caspase 9, activated by prolonged mitotic arrest, is not activated and is not required for apoptosis under our experimental conditions. P53 upregulates expression of cFLIPL, a protein that blocks caspase 8 activation. cFLIPL levels are lower in cells lacking p53 and these levels are reduced to a greater extent after stathmin depletion. Expression of FLAG-tagged cFLIPL in p53-deficient cells rescues them from apoptosis triggered by stathmin depletion or CDK1 inhibition during G2. These data indicate that a cell cycle delay in G2 activates caspase 8 to initiate apoptosis specifically in p53-deficient cells.