Liaisons between Survivin and Plk1 during Cell Division and Cell Death

Survivin and Plk1 kinase are important mediators of cell survival that are required for chromosome alignment, cytokinesis, and protection from apoptosis. Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis. Moreover, the expression of both survivin and Plk1 is deregulated in cancer. Given these similarities, we speculated that these two proteins may cooperate during mitosis and/or in cell death pathways. Here we report that survivin and Plk1 interact during mitosis and that Plk1 phosphorylates survivin at serine 20. Importantly, we find that overexpression of a non-phosphorylatable version, S20A, is unable to correct chromosomes connected to the spindle in a syntelic manner during prometaphase and allows cells harboring these maloriented chromosomes to enter anaphase, evading the spindle tension checkpoint. By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein. Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL. Together, these data suggest that phosphorylation of survivin at Ser²⁰ by Plk1 kinase is essential for accurate chromosome alignment and cell proliferation but is dispensable for its anti-apoptotic activity in cancer cells.     

Sfrp Controls Apicobasal Polarity and Oriented Cell Division in Developing Gut Epithelium

Epithelial tubular morphogenesis leading to alteration of organ shape has important physiological consequences. However, little is known regarding the mechanisms that govern epithelial tube morphogenesis. Here, we show that inactivation of Sfrp1 and Sfrp2 leads to reduction in fore-stomach length in mouse embryos, which is enhanced in the presence of the Sfrp5 mutation. In the mono-cell layer of fore-stomach epithelium, cell division is normally oriented along the cephalocaudal axis; in contrast, orientation diverges in the Sfrps-deficient fore-stomach. Cell growth and apoptosis are not affected in the Sfrps-deficient fore-stomach epithelium. Similarly, cell division orientation in fore-stomach epithelium diverges as a result of inactivation of either Stbm/Vangl2, an Fz/PCP component, or Wnt5a. These observations indicate that the oriented cell division, which is controlled by the Fz/PCP pathway, is one of essential components in fore-stomach morphogenesis. Additionally, the small intestine epithelium of Sfrps compound mutants fails to maintain proper apicobasal polarity; the defect was also observed in Wnt5a-inactivated small intestine. In relation to these findings, Sfrp1 physically interacts with Wnt5a and inhibits Wnt5a signaling. We propose that Sfrp regulation of Wnt5a signaling controls oriented cell division and apicobasal polarity in the epithelium of developing gut.     

The Active Oxygen Response of Cell Suspensions to Incompatible Bacteria Is Not Sufficient to Cause Hypersensitive Cell Death

The inoculation of tobacco (Nicotiana tabacum L.) suspension cells with bacterial pathogens that elicit the hypersensitive response (HR) in leaves has been shown to elicit production of active oxygen. This response occurs in two phases, the second of which occurs 1 to 3 h after bacterial addition and is unique to HR-causing interactions. The relationship between the phase II active oxygen response and the HR was characterized using Pseudomonas syringae pv syringae and P. fluorescens (pHIR11), which contains a cosmid clone of the hrp/hrm region from P. syringae pv syringae. TnphoA mutations in complementation groups II through XIII of the hrp cluster blocked the phase II active oxygen response, whereas mutations in the group I hrmA locus did not affect phase II. Despite the normal active oxygen response, bacteria with mutations in the hrmA region did not cause the HR in intact tobacco leaves nor did they induce hypersensitive cell death in cell suspensions. The data indicate that the bacteria do not require the hrmA region to elicit active oxygen production, but a full and intact hrp/hrm region is required to elicit hypersensitive cell death. Therefore, the phase II active oxygen response does not directly cause hypersensitive cell death nor is the response itself sufficient to trigger the HR.     

Bax Deletion Further Orders the Cell Death Pathway in Cerebellar Granule Cells and Suggests a Caspase-independent Pathway to Cell Death

Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 μM NMDA. Within 2 h after switching to 5 mM K⁺, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax −/− neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K⁺ deprivation. Wild-type granule cells in 5 mM K⁺ increased cleavage of DEVD–aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.     

Transfection of N-Methyl-d-Aspartate Receptors in a Nonneuronal Cell Line Leads to Cell Death

Abstract: Neurons grown in culture die when they are exposed to high concentrations (0.1–1 m M ) of the neurotransmitter l -glutamate. A similar phenomenon may occur in the mammalian brain during ischemia and other injuries that cause excessive glutamate release. Activation of N -methyl- d -aspartate (NMDA) receptors and the consequent Ca²⁺ influx are thought to play a critical role in the process of neuronal toxicity. Events subsequent to the Ca²⁺ influx are not well understood. We have discovered that nonneuronal kidney cells expressing NMDA receptors after DNA transfection undergo cell death unless they are protected by drugs that block the NMDA receptor ion channel. Furthermore, transfected cells expressing a mutated NMDA receptor that conducts less Ca²⁺ are less vulnerable to cell death. In addition, we find that even though several active forms of NMDA receptors can be synthesized in these cells after transfection with different cloned subunits, not all receptor types are equally toxic. These experiments suggest that Ca²⁺ influx through NMDA channels may be toxic to nonneuronal cells and that the NMDA receptor expression may be the major neuron-specific component of excitotoxicity.     

Trypanosoma cruzi Response to Sterol Biosynthesis Inhibitors: Morphophysiological Alterations Leading to Cell Death

The protozoan parasite Trypanosoma cruzi displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of T. cruzi cells following treatment with the sterol biosynthesis inhibitors (SBIs) ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC₅₀/72 h) or killing all cells within 24 hours (EC₁₀₀/24 h). Incubation with inhibitors at the EC₅₀/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC₅₀/72 h. By contrast, treatment with SBIs at the EC₁₀₀/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP), culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the “point of no return” in the cell death cascade, and propose a model for the necrotic cell death of T. cruzi. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in T. cruzi. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite.     

c-Myc: Where Death and Division Collide

c-Myc is intimately involved in cell proliferation. However, inappropriate activation of c-Myc may also promote or sensitize cells to apoptosis. This is well established for several cell types and has been reinforced by a recent report in Cell Death and Differentiation by de Alboran and colleagues, who report that c-Myc-deficient B cells are resistant to several apoptotic stimuli, supporting a role for c-Myc in modulating B lymphocyte cell death. Here, we integrate these findings into the current picture of how c-Myc participates in cell death control.     

Involvement of Yeast HSP90 Isoforms in Response to Stress and Cell Death Induced by Acetic Acid

Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2α by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress.     

Commitment to Division in Ciliate Cell Cycles

Near the end of the cell cycle, ciliates commit irreversibly to cell division. The point of commitment occurs at the time of oral polykinetid assembly and micronuclear anaphase. The commitment is a checkpoint which requisites a threshold cell mass/ DNA ratio and stomatogenesis. It is also a physiological transition point, involving cdk protein kinases similar to those of other eukaryotes. Both P34 kD and P36 kD kinases, similar to the S. pombe cdc2 kinases, have been described to have activity as monomers. Subsequent to commitment to division, dramatic cytoskeletal modifications occur for separation of organelles, cortex morphogenesis and cytokinesis. Numerous mutants affecting cytoskeletal function associated with the division process have been obtained in several species. Of these, only the ccl mutant in Paramecium affects cell cycle progression prior to commitment to division. The material reviewed is used to speculate about the mechanisms of regulation of pre-fission morphogenesis and cell division related processes in ciliates.     

Epigenetics and Cell Death: DNA Hypermethylation in Programmed Retinal Cell Death

Background

Vertebrate genomes undergo epigenetic reprogramming during development and disease. Emerging evidence suggests that DNA methylation plays a key role in cell fate determination in the retina. Despite extensive studies of the programmed cell death that occurs during retinal development and degeneration, little is known about how DNA methylation might regulate neuronal cell death in the retina.

Methods

The developing chicken retina and the rd1 and rhodopsin-GFP mouse models of retinal degeneration were used to investigate programmed cell death during retinal development and degeneration. Changes in DNA methylation were determined by immunohistochemistry using antibodies against 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).

Results

Punctate patterns of hypermethylation paralleled patterns of caspase3-dependent apoptotic cell death previously reported to occur during development in the chicken retina. Degenerating rd1 mouse retinas, at time points corresponding to the peak of rod cell death, showed elevated signals for 5mC and 5hmC in photoreceptors throughout the retina, with the most intense staining observed in the peripheral retina. Hypermethylation of photoreceptors in rd1 mice was associated with TUNEL and PAR staining and appeared to be cCaspase3-independent. After peak rod degeneration, during the period of cone death, occasional hypermethylation was observed in the outer nuclear layer.

Conclusion

The finding that cell-specific increases of 5mC and 5hmC immunostaining are associated with the death of retinal neurons during both development and degeneration suggests that changes in DNA methylation may play a role in modulating gene expression during the process of retinal degeneration. During retinal development, hypermethylation of retinal neurons associates with classical caspase-dependent apoptosis as well as caspase-3 independent cell death, while hypermethylation in the rd1 mouse photoreceptors is primarily associated with caspase-3 independent programmed cell death. These findings suggest a previously unrecognized role for epigenetic mechanisms in the onset and/or progression of programed cell death in the retina.