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Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death. [ABSTRACT FROM AUTHOR]

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features. [ABSTRACT FROM AUTHOR]

Different types of cell death are often defined by morphological criteria, without a clear reference to precise biochemical mechanisms. The Nomenclature Committee on Cell Death (NCCD) proposes unified criteria for the definition of cell death and of its different morphologies, while formulating several caveats against the misuse of words and concepts that slow down progress in the area of cell death research. Authors, reviewers and editors of scientific periodicals are invited to abandon expressions like ‘percentage apoptosis’ and to replace them with more accurate descriptions of the biochemical and cellular parameters that are actually measured. Moreover, at the present stage, it should be accepted that caspase-independent mechanisms can cooperate with (or substitute for) caspases in the execution of lethal signaling pathways and that ‘autophagic cell death’ is a type of cell death occurring together with (but not necessarily by) autophagic vacuolization. This study details the 2009 recommendations of the NCCD on the use of cell death-related terminology including ‘entosis’, ‘mitotic catastrophe’, ‘necrosis’, ‘necroptosis’ and ‘pyroptosis’.Cell Death and Differentiation (2009) 16, 3–11; doi:10.1038/cdd.2008.150; published online 10 October 2008 [ABSTRACT FROM AUTHOR]

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21Waf1 (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.Oncogene (2007) 26, 6954–6958. doi:10.1038/sj.onc.1210516; published online 7 May 2007 [ABSTRACT FROM AUTHOR]

The Bcr-Abl fusion protein drives leukemogenesis and can render leukemia cells resistant to conventional chemotherapy. Geldanamycin (GA), a drug which destabilizes Hsp90-associated proteins, depletes cells of Bcr-Abl, an Hsp90 client, but not of Abl. Both HL60 cells transfected with Bcr-Abl and naturally Ph1-positive K562 leukemia cells are resistant to most cytotoxic drugs, but were found to be sensitive to GA. Furthermore, GA sensitized Bcr-Abl-expressing cells to doxorubicin (DOX) and paclitaxel (PTX). In contrast, in parental HL60 cells, 90 nM GA inhibited PARP cleavage, nuclear fragmentation, and cell death caused by 500 ng/ml DOX. Like GA, STI 571 (an inhibitor of the Abl kinase) sensitized Bcr-Abl-expressing cells to DOX. Unlike GA, STI 571 did not antagonize the cytotoxic effects of DOX in parental HL60 cells. These results indicate that sensitization of Bcr-Abl-expressing cells, but not desensitization of HL60 cells, depends on inhibition of Bcr-Abl. Thus, GA differentially affects leukemia cells depending on their Bcr-Abl expression and selectively increases apoptosis in Bcr-Abl-expressing cells. [ABSTRACT FROM AUTHOR]

Recent evidence indicates that anti-apoptotic functions of BcI-2 can be regulated by its phosphorylation. According to the 'mitotic arrest-induced' model, multi-site phosphorylation of the BcI-2 loop domain is followed by cell death. In contrast, in cytokine-dependent cell lines, cytokines mediate phosphorylation of BcI-2 on S70, preventing apoptosis. As discussed in this review, these models are not mutually exclusive but reflect different cellular contexts. During mitotic arrest, signal transduction is unique and is fundamentally different from classical mitogenic signaling, since the nucleus membrane is dissolved, gene expression is reduced, and numerous kinases and regulatory proteins are hyperphosphorylated. Hyperphosphorylation of BcI-2 mediated by paclitaxel and other microtubule-active drugs is strictly dependent on targeting microtubules that in turn cause mitotic arrest. In addition to serine-70 (S70), microtubule-active agents promote phosphorylation of S87 and threonine-69 (T69), inactivating BcI-2. A major obstacle for identification of the mitotic BcI-2 kinase(s) is that inhibition of putative kinase(s) by any means (dominant-negative mutants, antisense oligonucleotides, pharmacological agents) may arrest cycle, preventing mitosis and BcI-2 phosphorylation. The role of BcI-2 phosphorylation in cell death is discussed. [ABSTRACT FROM AUTHOR]

Many chemotherapeutic agents induce apoptosis in tumor cells, but killing of normal cells remains a major obstacle. Development of multidrug resistance further limits chemotherapy in cancer. Here, I show that multidrug resistance can be exploited for selective killing of multidrug-resistant cells by a combination of an apoptosis-inducing agent that is not a substrate of either Pgp or MRP (e.g. flavopiridol) with a caspase inhibitor that is a substrate (e.g. Z-DEVD-fmk). In normal cells, treatment with caspase inhibitors prevented PARP cleavage, nuclear fragmentation, and cell death caused by flavopiridol or epothilone B. In contrast, Pgp- and MRP-expressing cells were not rescued by caspase inhibitors. Furthermore, reversal of drug resistance renders Pgp cells sensitive to caspase inhibitors abolishing therapeutic advantage. Thus, caspase inhibitors, that are inactive in multidrug-resistant cells, protect normal but not multidrug-resistant cells against chemotherapy, permitting selective eradication of multidrug-resistant cells. Clinical application of this approach may diminish the toxic side-effects of chemotherapy in patients with multidrug-resistant tumors. [ABSTRACT FROM AUTHOR]

Preclinical models for the identification of prostate cancer chemoprevention agents are lacking. Based upon the notion that clinically useful chemoprevention agents should exhibit selective activity against early stage disease, studies were undertaken to assess whether chemoprevention agents selectively inhibited the growth of early stage prostate cancer, as compared to late stage cancer. First, a series of cell and molecular studies were performed, which, when taken together, validated the use of a panel of prostate cell lines as a model of the different stages of carcinogenesis. Next, therapeutic responsiveness to ten different cytotoxic or chemoprevention agents was evaluated. Chemoprevention agents exhibited selective activity against normal and early transformed prostate tissue, whereas cytotoxic agents were non-specific. Selective activity against early versus advanced prostate cancer cells is identified as a potential screening method for chemoprevention agents.Prostate Cancer and Prostatic Diseases (2001) 4, 81–91 [ABSTRACT FROM AUTHOR]

Depsipeptide, FR901228, a novel cyclic peptide inhibitor of histone deacetylase with a unique cytotoxicity profile is currently in phase I clinical trials. Here we demonstrate that, in addition to G2/M arrest, FR901228 causes G1 arrest with Rb hypophosphorylation. In vitro kinase assays demonstrated no direct inhibition of CDK activity, however, an inhibition was observed in CDKs extracted from cells exposed to FR901228. Cyclin D1 protein disappeared between 6 and 12 hours after treatment with FR901228, whereas cyclin E was upregulated. While it did not induce wt p53, FR901228 did induce p21[SUPWAF1/CIP1] in a p53-independent manner. Cell clones lacking p21 were not arrested in G1 phase, but continued DNA synthesis and were arrested in G2/M phase following FR901228 treatment. Finally, FR901228 blunted ERK-2/MAPK activation by EGF whereas early signal transduction events remained intact since overall cellular tyrosine phosphorylation after EGF stimulation was unaffected. Thus, FR901228, while not directly inhibiting kinase activity, causes cyclin D1 downregulation and a p53-independent p21 induction, leading to inhibition of CDK and dephosphorylation of Rb resulting in growth arrest in the early G1 phase. In contrast to the G1 arrest, the G2/M arrest is p21-independent, but is associated with significant cytotoxicity. [ABSTRACT FROM AUTHOR]

Though the term apoptosis was originated in pathology and developmental biology as an alternative to necrosis, the tissue necrosis with inflammation is irrelevant to cell culture conditions where apoptosis is mostly studied. Furthermore, no one single morphological feature is either necessary or sufficient to define apoptosis. The emerging biochemical definition, a cell death with caspase activation, allows the distinction of alternative forms of cell death. Thus, inhibition of caspases delays but does not prevent cell death. Slow cell death without caspase activation may nevertheless be associated with DNA fragmentation. Oncogenic Ras, Raf, and mitogen-activated kinases inhibit apoptosis by affecting the cytochrome C/caspase-9 pathway but may arrest growth and cause slow cell death with delayed DNA fragmentation. Such 'slow' cell death without caspase activation is often caused by chemotherapeutic drugs. Whether a cell will undergo apoptosis or slow death depends not only on a chemotherapeutic agent but also on the readiness of cellular caspases. Therefore, one can distinguish apoptosis-prone (eg leukemia) vs apoptosis-resistant cells. Cell susceptibilities to spontaneous, starvation-induced and drug-induced apoptosis are correlated and characterize an apoptosis-prone phenotype. Finally, distinction of slow cell death allows rephrasing of a question regarding the goal of cancer therapy: apoptosis vs slow cell death, or cancer cell-selectivity regardless of the mode of cell death. [ABSTRACT FROM AUTHOR]

Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>>ALLN>>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition. [ABSTRACT FROM AUTHOR]

Drug resistance is a well recognized problem in cancer therapy. Despite the current dogma that drug resistance is always an obstacle for treatment, here I show that it provides opportunities for selective protection of non-resistant cells with killing of drug-resistant cancer cells. According to the proposed 'two-drug' strategy, the first drug should be ineffective against a target drug-resistant cell (ie the drug is a substrate of MRP or Pgp pumps). In addition, it must be cytostatic but not cytotoxic. The second drug, which is applied in sequence, must be a cycle-dependent apoptotic drug to which the target cell is not cross-resistant. Thus, low doses of adriamycin, etoposide and actinomycin D, used as the first drugs, were cytostatic to parental HL60 cells. Therefore, these drugs precluded Bcl-2/Raf-1 phosphorylation, PARP cleavage and cell death which are otherwise induced by paclitaxel, a mitosis-selective apoptotic drug for HL60 cells. In contrast, HL60/ADR cells which express MRP, a transporter which pumps out the first drugs from a cell, were insensitive to the first drugs and therefore readily underwent apoptosis following the second drug. This strategy also allowed a selective killing of HL60/TX cells which express MDR-1, with the only difference being that the second drug, paclitaxel, was substituted for epothilones, non-Pgp substrates. Lack of protection by the first drug, a Pgp substrate, resulted in HL60/TX killing by the second drug, whereas parental HL-60 cells were fully protected. Therefore, drug resistant cells can be selectively killed by a combination of drugs not killing sensitive cells. Lack of toxicity against normal cells will be clinically translated in reduction of adverse side-effects of chemotherapy against drug-resistant malignancies. [ABSTRACT FROM AUTHOR]

Raf-1 activation and Bcl-2 hyperphosphorylation following treatment with paclitaxel (Taxol) or other microtubule-active drugs is associated with mitotic arrest. Here we show that microtubule-active drugs do not activate the mitogen-activated protein kinase (MAPK) pathway in leukemia cells. PD98059, a MEK inhibitor, and SB202190, a p38 MAP kinase inhibitor, do not abrogate Bcl-2 phosphorylation nor apoptosis. Simultaneously with PARP cleavage, paclitaxel induces cleavage of Bcl-2 protein yielding a potentially pro-apoptotic 22 kDa product. In comparison, the stimulation of Raf-1 by phorbol ester (TPA) activates the MAPK pathway, causes MAPK-dependent p21WAF1/CIP1 induction, Rb dephosphorylation and growth arrest without Bcl-2 phosphorylation or apoptosis. Like TPA, cAMP induces p21WAF1/CIP1 but does not cause Bcl-2 phosphorylation. MEKK1 and Ras, upstream activators of JNK and ERK MAPK, also fail to induce Bcl-2 hyperphosphorylation. Although Lck tyrosine kinase has been recently implicated in Raf-1 activation during mitotic arrest, microtubule-active drugs induce Raf-1/Bcl-2 hyperphosphorylation and apoptosis in a Lck-deficient Jurkat cells. Therefore, microtubule-active drugs induce apoptosis which is associated with Raf-1 and Bcl-2 phosphorylation and Bcl-2 cleavage but is independent of the MAPK pathway. In contrast, TPA-activated MAPK pathway causes p21WAF1/CIP1-dependent growth arrest without apoptosis. [ABSTRACT FROM AUTHOR]

Examines the role of CDK2 in the G1/S transition. Discussion of Bcr-Abl's capability in blocking several steps of the apoptotic cascade; Description of the inhibition of Bcr-Abl's influence in eliminating a driving force in the cell cycle, such as Bcr-Abl-activated CDK2; Influence of Gleevec on the growth arrest and cell death in Bcr-Abl-addicted leukemias.