A variety of key events in apoptosis focus on mitochondria, including the release of caspase activators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro- and antiapoptotic Bcl-2 family proteins. The different signals that converge on mitochondria to trigger or inhibit these events and their downstream effects delineate several major pathways in physiological cell death.

https://science.sciencemag.org/content/sci/281/5381/1309.full.pdf

Mitochondria and apoptosis

Mitogen-dependent progression through the first gap phase (G1) and initiation of DNA synthesis (S phase) during the mammalian cell division cycle are cooperatively regulated by several classes of cyclin-dependent kinases (CDKs) whose activities are in turn constrained by CDK inhibitors (CKIs). CKIs that govern these events have been assigned to one of two families based on their structures and CDK targets. The first class includes the INK4 proteins (inhibitors of CDK4), so named for their ability to specifically inhibit the catalytic subunits of CDK4 and CDK6. Four such proteins [p16 (Serrano et al. 1993), p15 (Hannon and Beach 1994), p18 (Guan et al. 1994; Hirai et al. 1995), and p19 (Chan et al. 1995; Hirai et al. 1995)] are composed of multiple ankyrin repeats and bind only to CDK4 and CDK6 but not to other CDKs or to D-type cyclins. The INK4 proteins can be contrasted with more broadly acting inhibitors of the Cip/Kip family whose actions affect the activities of cyclin D-, E-, and A-dependent kinases. The latter class includes p21 (Gu et al. 1993; Harper et al. 1993; El-Deiry et al. 1993; Xiong et al. 1993a; Dulic et al. 1994; Noda et al. 1994), p27 (Polyak et al. 1994a,b; Toyoshima and Hunter 1994), and p57 (Lee et al. 1995; Matsuoka et al. 1995), all of which contain characteristic motifs within their amino-terminal moieties that enable them to bind both to cyclin and CDK subunits (Chen et al. 1995, 1996; Nakanishi et al. 1995; Warbrick et al. 1995; Lin et al. 1996; Russo et al. 1996). Based largely on in vitro experiments and in vivo overexpression studies, CKIs of the Cip/Kip family were initially thought to interfere with the activities of cyclin D-, E-, and A-dependent kinases. More recent work has altered this view and revealed that although the Cip/Kip proteins are potent inhibitors of cyclin Eand A-dependent CDK2, they act as positive regulators of cyclin Ddependent kinases. This challenges previous assumptions about how the G1/S transition of the mammalian cell cycle is governed, helps explain some enigmatic features of cell cycle control that also involve the functions of the retinoblastoma protein (Rb) and the INK4 proteins, and changes our thinking about how either p16 loss or overexpression of cyclin D-dependent kinases contribute to cancer. Here we focus on the biochemical interactions that occur between CKIs and cyclin Dand E-dependent kinases in cultured mammalian cells, emphasizing the manner by which different positive and negative regulators of the cell division cycle cooperate to govern the G1-to-S transition. To gain a more comprehensive understanding of the biology of CDK inhibitors, readers are encouraged to refer to a rapidly emerging but already extensive literature (for review, see Elledge and Harper 1994; Sherr and Roberts 1995; Chellappan et al. 1998; Hengst and Reed 1998a; Kiyokawa and Koff 1998; Nakayama 1998; Ruas and Peters 1998).

/pdf/cdk-inhibitors-positive-and-negative-regulators-of-g1-phase-4awlfdse3n.pdf

CDK inhibitors: positive and negative regulators of G1-phase progression

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells (G(0)), makes it an excellent marker for determining the so-called growth fraction of a given cell population. In the first part of this study, the term proliferation marker is discussed and examples of the applications of anti-Ki-67 protein antibodies in diagnostics of human tumors are given. The fraction of Ki-67-positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of the disease. The best-studied examples in this context are carcinomas of the prostate and the breast. For these types of tumors, the prognostic value for survival and tumor recurrence has repeatedly been proven in uni- and multivariate analysis. The preparation of new monoclonal antibodies that react with the Ki-67 equivalent protein from rodents now extends the use of the Ki-67 protein as a proliferation marker to laboratory animals that are routinely used in basic research. The second part of this review focuses on the biology of the Ki-67 protein. Our current knowledge of the Ki-67 gene and protein structure, mRNA splicing, expression, and cellular localization during the cell-division cycle is summarized and discussed. Although the Ki-67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear. There are indications, however, that Ki-67 protein expression is an absolute requirement for progression through the cell-division cycle.

The Ki‐67 protein: From the known and the unknown

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Filling a GAP(DH) in caspase-independent cell death.

Apoptosis is a genetically driven form of cell death that results in systematic dismantling of the dying cell. Apoptotic cell death, a process critical for development and tissue homeostasis in multicellular organisms, is carried out by a family of proteases known as caspases. Caspases that lie at the start of apoptotic pathways are known as initiator or apical caspases. They in turn activate executioner caspases, which go on to cleave the various cellular substrates that package the cell for elimination. Apical caspases are first activated by adaptor proteins that are typically targeted by upstream apoptotic signals. Although the mechanism of adaptor protein activation is in some cases controversial, considerable characterization has been reported for the vertebrate Apaf-1 adaptor protein that is activated by cytochrome c. Cytosolic cytochrome c, released from the mitochondria during cell death binds to and induces oligomerization of Apaf-1 into heptamers, which in turn recruit and activate caspase 9. In vertebrate cells, mitochondrial events of apoptosis, including cytochrome c release, are regulated by the Bcl-2 family. Antiapoptotic Bcl-2 family members (Bcl-2, Bcl-xL, Mcl-1) inhibit release of cytochrome c, whereas proapoptotic Bcl-2 family members (Bax, Bak, Bid, Bim Bad) promote cytochrome c release (reviewed by Cory et al. and Danial and Korsmeyer). In Drosophila melanogaster, the regulation of caspase activation is not fully understood. Although a Drosophila homolog of Apaf-1, known variously as Drosophila Apaf-1related killer (Dark), homolog of Apaf-1 and Ced4 (Hac-1) or Drosophila Apaf-1 (dApaf-1) has been characterized and is known to promote activation of the initiator caspase Dronc, it is not known how or even if Dark is directly regulated. Reports from several laboratories have shown that Dark binds to cytochrome c and that cytochrome c can induce the formation of a Dark apoptosome in vitro, although there have not yet been any reports documenting release of cytochrome c from Drosophila mitochondria in response to apoptotic stimuli. Additionally, a recent report suggests that cytochrome c is neither present within nor required for formation of Dark/Dronc complexes. In addition, although Drosophila cytochrome c isoforms can induce cell death upon ectopic expression in vertebrates, depletion of cytochrome c has no apparent effect on cell death in the fly. Moreover, reconstituted apoptosis in fly cell extract does not require mitochondrial factors. However, there is evidence that caspase activation during spermatid differentiation is dependent on cytochrome c, but it is not known if this reflects an ability of cytochrome c to bind and promote Dark activation. In addition to controversy over the role of the mitochondria in fly cell death, it is also unclear how the two Bcl2 family members in Drosophila, Buffy (dBorg-2) and Debcl (Drob-1/dBorg-1/dBok) regulate cell death. Buffy, thought to behave as an antiapoptotic Bcl-2 family member, inhibits cell death during development as well as in response to g irradiation. Debcl, a proapoptotic Bcl-2 family member mediates cell death during development, during UV irradiation, and when ectopically expressed. A specific function for Buffy or Debcl has not been definitively shown and it is not clear if the Drosophila Bcl-2 family members act on mitochondria, like their vertebrate counterparts. Thus, although sequence homology can be informative, it may be misleading; it is not yet clear whether sequence relatedness is at all indicative of functional similarities between fly and vertebrate Bcl2 family members.

Multifunctional reaper: sixty-five amino acids of fury.

Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules In Drosophila, the ubiquitin–proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1 Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated Here we report the identification of a DIAP1-directed DUB using two complementary approaches First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which we named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation

/pdf/the-deubiquitinating-enzyme-dubai-stabilizes-diap1-to-2ytlitjhga.pdf

The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis

EMBO J 29 14, 2407–2420 (2010); published online June042010 [PMC free article] [PubMed]

Microtubule poisons induce mitotic arrest that leads to apoptotic cell death if not resolved in a timely manner, but the mechanisms that directly link this cell cycle arrest to apoptosis have been elusive. In this issue of The EMBO Journal, Clarke and colleagues show that Mcl-1, an anti-apoptotic Bcl-2 family protein, is phosphorylated by the mitotic kinase CDK1/cyclin B1. This targets the protein for degradation by anaphase-promoting complex/cyclosome (APC/C)-mediated ubiquitination, in a manner such that only prolonged arrest allows sufficient Mcl-1 phosphorylation and degradation to trigger apoptosis. Thus, the APC/C, a major effector of the spindle assembly checkpoint (SAC), not only ensures cell cycle arrest upon spindle disruption, but promotes cell death when the duration of mitotic arrest is too long.

Apoptotic cell death proceeds through a series of signalling events that bring about the dismantling of cellular components by a family of cysteine proteases, the caspases. In particular, genotoxic and/or cytotoxic stress promotes the release of cytochrome c, a component of the electron transport chain in the mitochondria, to the cytoplasm, which in turn triggers activation of the initiator and executioner caspases, caspase-9 and caspase-3, respectively. It is well recognized that a failure in cell cycle progression, such as prolonged mitotic arrest, can induce apoptosis by triggering mitochondrial cytochrome c release. When a cell encounters a defect in mitotic spindle assembly (as when cells are treated with microtubule poisons), entry into anaphase is inhibited by the SAC that suppresses APC/C-mediated cyclin B1 degradation until metaphase can be completed; if the spindle defect is unrepairable, apoptotic cell death ensues. Cyclin B1 degradation is slowed, but not halted, during prolonged mitotic arrest because of residual APC/CCdc20 activity. As a result, when CDK1 activity drops below the threshold required for maintenance of mitosis, cells eventually exit M phase with a defect in chromosome segregation. Thus, mitotic cell death seems to be a preventive mechanism that avoids the creation of carcinogenic aneuploids.

Harley et al (2010) now show that the degradation of the anti-apoptotic Bcl-2 family member Mcl-1 has an important function in such mitotic cell death. Cytochrome c release is regulated by the balance of pro- and anti-apoptotic Bcl-2 family proteins. Owing to its very short half-life (<0.5 h), Mcl-1 protein levels, and its consequent contribution to cell survival, can be rapidly tuned by changes in protein stability. Polyubiquitination and proteasomal degradation of Mcl-1 in interphase has been attributed to the action of two E3 ligases, Mule and SCFβ-TrCP; this can be counter-acted by the deubiquitinase USP9X (Zhong et al, 2005; Ding et al, 2007; Schwickart et al, 2010). Harley et al (2010) add another layer of Mcl-1 stability control, showing that APC/CCdc20 is a mitosis-specific E3 ligase for Mcl-1, which prompts the destruction of Mcl-1 during prolonged mitotic arrest. As is true for multiple APC/C substrates, the interaction between Mcl-1 and the APC/C activator Cdc20 is mediated through a D-box motif. Unlike cyclin B, but similar to the situation for cyclin A, APC/C-mediated Mcl-1 ubiquitination is not inhibited by the SAC. Further, APC/CCdc20-mediated ubiquitination of Mcl-1 is unusual in that it requires earlier phosphorylation of Mcl-1 by CDK1/cyclin B1. Thus, Mcl-1 carrying either a phospho-site or a D-box mutation exhibits marked stability during prolonged mitosis. The authors also show that expression of the mutant Mcl-1, but not wild-type protein, renders cells resistant to apoptotic cell death induced by mitotic arrest, indicating that the APC/CCdc20-mediated degradation of Mcl-1 is critical for mitotic cell death. As Cdc20-Mcl-1 interactions do not seem to be modulated by this phosphorylation, Harley et al speculate that the ability of the APC/C to act on this complex may be phospho-dependent or that a deubiquitinase countering Mcl-1 polyubiqutiylation may be antagonized by Mcl-1 phosphorylation. A further important facet of the proposed model is a timing mechanism. During a normal mitosis, the transient phosphorylation of Mcl-1 by CDK1/cyclin B1 is insufficient to drive degradation of the majority of Mcl-1 before cyclin B1 degradation and loss of CDK1 activity. Thus, cells are not unduly susceptible to apoptosis if mitosis proceeds normally. However, on prolonged SAC arrest with sustained CDK1/cyclin B1 activity, the accrued phosphorylation promotes sufficient degradation to drop Mcl-1 below the threshold necessary to sustain cell viability. This ensures that only prolonged arrest induces apoptosis.

The mechanism of mitotic cell death has long been unclear. A recent study showed that Bcl-xL and Bcl-2 are phosphorylated by CDK1/cyclin B1 and inhibited in mitosis (Terrano et al, 2010). However, it has also been shown that CDK1 activity is required to protect cells from apoptosis in mitosis (O'Connor et al, 2002). How can we reconcile these apparently paradoxical results? In contrast to prolonged mitotic arrest, cells in normal mitosis exhibit resistance to apoptosis. For instance, during M phase, caspase-2 and caspase-9 are inhibited by CDK1/cyclin B1-mediated phosphorylation, thus preventing apoptosis (Allan and Clarke, 2007; Andersen et al, 2009).

The findings by Harley et al (2010) provide new insight into the CDK1/cyclin B1-mediated regulation of pro- and anti-apoptotic proteins in mitosis. It is suggested that during mitosis in which high CDK1/cyclin B1 activity is maintained, the anti-apoptotic function of Bcl-xL and Bcl-2 is suppressed, whereas Mcl-1 is gradually degraded (Figure 1A). Concomitantly, CDK1/cyclin B1 inhibits the activation of the initiator caspase-2 and caspase-9, thereby preventing the full initiation of the apoptotic cascade (Figure 1A). When mitotic arrest is protracted, the loss of Mcl-1 ultimately elicits the release of cytochrome c from the mitochondria (Figure 1B). Interestingly, the authors show that during mitotic arrest, Mcl-1 degradation precedes the dephosphorylation of caspase-9. In that a non-phosphorylatable mutant of caspase-2 or caspase-9 can enhance sensitivity to apoptosis in mitotic cells (Allan and Clarke, 2007; Andersen et al, 2009), it is likely that a signal distinct from Mcl-1 degradation also takes part in mitotic cell death through the dephosphorylation of the caspases. Lastly, it is known that some cancer cells overexpress Mcl-1, whereas others do not (Warr and Shore, 2008; Schwickart et al, 2010). It will be interesting to compare the efficacy of microtubule-directed chemotherapeutics in cancers expressing varying levels of Mcl-1 in that this may serve as a novel biomarker to predict patient responsiveness to these agents.



Figure 1

Model for mitosis-induced apoptosis. (A) In normal mitosis, CDK1/cyclin B1-mediated phosphorylation (P) inhibits pro- and anti-apoptotic proteins, including Bcl-xL, Bcl-2, caspase-2 (C2) and caspase-9 (C9), whereas it causes the APC/CCdc20-mediated ubiquitination ...

/pdf/stalling-in-mitosis-and-releasing-the-apoptotic-brake-23dj2xfpbx.pdf

Stalling in mitosis and releasing the apoptotic brake

Study question Can host fertility be rescued by grafting of a fragment of a healthy ovary soon after chemotherapy? Summary answer We found that grafting a green fluorescent protein (GFP)-positive fragment from a healthy isogenic ovary to the left ovary of a chemo-treated host rescued function and fertility of the grafted host ovary, and resulted in the production of host-derived offspring as late as the sixth litter after chemotherapy (CTx) treatment, whereas none of the ungrafted controls produced a second litter. What is known already In women and girls undergoing chemotherapy, infertility and premature ovarian failure are frequent outcomes. There are accumulating reports of improved endocrine function after autotransplantation of an ovarian fragment, raising the possibility that the transplant is beneficial to the endogenous ovary. Study design, size, duration We first established a CTx treatment regimen that resulted in the permanent loss of fertility in 100% of female mice of the FVB inbred strain. We grafted an isogenic ovary fragment from a healthy female homozygous for a GFP transgene to the left ovary of 100 CTx-treated hosts, and compared fertility to 39 ungrafted controls in 6 months of continuous matings, using GFP to distinguish offspring derived from the graft, and those derived from the host. Participants/materials, setting, methods Immunofluoresece and western blot analysis of 39 treated ovaries during and 15 days after CTx treatment revealed elevated apoptosis, rapid loss of granulosa cells and an increased recruitment of growing follicles. Using immunofluorescence and confocal imaging, we tracked the outcome of the grafted tissue over 4 months and its effect on the adjacent and contralateral ovary of the host. Main results and the role of chance Fifty-three percent of grafted females produced a second litter whereas none of the ungrafted females produced a second litter. The likelihood that this could occur by chance is very low (P Limitations, reasons for caution These results are shown only in mice, and whether or how they might apply to chemotherapy patients subjected to different CTx regimens is not yet clear. Wider implications of the findings Our experiments prove that rescue of a chemo-treated ovary is possible, and establish a system to investigate the mechanism of rescue and to identify the factors responsible with the long-term goal of developing therapies for preservation of ovarian endocrine function and fertility in women undergoing chemotherapy. Large scale data No large datasets were produced. Study funding/competing interests Duke University Medical Center Chancellor's Discovery Grant to BC; ESJ was supported by an NRSA 5F31CA165545; SK was supported by NIH RO1 GM08033; RWT was supported by the Duke University School of Medicine Ovarian Cancer Research Fellowship; XBM was supported by CONICYT. The authors have no conflicts of interest to declare.

/pdf/a-grafted-ovarian-fragment-rescues-host-fertility-after-50ntnx7fo4.pdf

Sally Kornbluth

Papers

Filling a GAP(DH) in caspase-independent cell death.

Multifunctional reaper: sixty-five amino acids of fury.

The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis

Stalling in mitosis and releasing the apoptotic brake

A grafted ovarian fragment rescues host fertility after chemotherapy