Marek Los

Apoptosis and cancer: mutations within caspase genes

The inactivation of programmed cell death has profound effects not only on the development but also on the overall integrity of multicellular organisms. Beside developmental abnormalities, it may lead to tumorigenesis, autoimmunity, and other serious health problems. Deregulated apoptosis may also be the leading cause of cancer therapy chemoresistance. Caspase family of cysteinyl-proteases plays the key role in the initiation and execution of programmed cell death. This review gives an overview of the role of caspases, their natural modulators like IAPs, FLIPs, and Smac/Diablo in apoptosis and upon inactivation, and also in cancer development. Besides describing the basic mechanisms governing programmed cell death, a large part of this review is dedicated to previous studies that were focused on screening tumours for mutations within caspase genes as well as their regulators. The last part of this review discusses several emerging treatments that involve modulation of caspases and their regulators. Thus, we also highlight caspase cascade modulating experimental anticancer drugs like cFLIP-antagonist CDDO-Me; cIAP1 antagonists OSU-03012 and ME-BS; and XIAP small molecule antagonists 1396–11, 1396–12, 1396–28, triptolide, AEG35156, survivin/Hsp90 antagonist shephedrin, and some of the direct activators of procaspase-3.

The role of caspases in development, immunity, and apoptotic signal transduction: lessons from knockout mice.

The rapid discovery of a great number of caspases, together with multiple control points of their activation, proceeds well ahead of our knowledge of their physiological roles within the organism. There are still major gaps, but recent gene targeting of caspases provides us with several new and fundamental aspects of their physiological functions. The fact that different lines of KO mice exhibit preferential apoptosis defects rather than a global suppression of apoptosis indicates that caspases play a largely nonredundant apoptotic role in a tissue- and stimulus-dependent manner. Furthermore, despite compelling evidence for a key role of Casp8 and Casp9, the restricted phenotype of both Casp8- and Casp9-deficient mice suggests that other apical caspases must exist regulating apoptotic processes. Casp10, for instance, which is very similar in its structure to Casp8, has been shown to be recruited to death receptors (13xIn vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Fernandes-Alnemri, T, Armstrong, R.C, Krebs, J, Srinivasula, S.M, Wang, L, Bullrich, F, Fritz, L.C, Trapani, J.A, Tomaselli, K.J, Litwack, G, and Alnemri, E.S. Proc. Natl. Acad. Sci. USA. 1996; 93: 7464–7469Crossref | PubMed | Scopus (653)See all References, 72xFas-associated death domain protein interleukin-1beta-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling. Vincenz, C and Dixit, V.M. J. Biol. Chem. 1997; 272: 6578–6583Crossref | PubMed | Scopus (239)See all References). Although fibroblasts from Casp8 null mice are almost completely resistant to death receptor–mediated apoptosis, it cannot be excluded that Casp10, having little importance in fibroblasts, exerts crucial functions in other cell types. In addition, since certain cell types such as embryonic fibroblasts from Apaf1 KO mice are still considerably sensitive to a variety of apoptosis inducers, it is very likely that other yet undiscovered key regulators exist. We also have to be aware that the restricted phenotype of most KO mice may underestimate the role of the targeted caspases, because single caspases may substitute other family members. A major obstacle of most KO mice is their prenatal lethality, which precludes manifestations of caspase functions in the adult organism. Therefore, in future research, conditional disruption in a cell type–specific manner or in certain developmental stages will be required to elucidate more precisely the in vivo functional significance of individual caspases in development, immune functions, and pathological forms of apoptosis.*To whom correspondence should be addressed (e-mail: kso@uni-muenster.de).†Present address: Division of Cell Biology, University of Munster, Rontgenweg 21, D-48149 Munster, Germany.

HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state.

This study demonstrates that human immunodeficiency virus type 1 (HIV‐1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HIV‐1 replication through activation of NF‐kappa B. In HeLa cells stably transfected with the HIV‐1 tat gene (HeLa‐tat cells), expression of the Tat protein enhanced both TNF‐induced activation of NF‐kappa B and TNF‐mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein. TNF‐mediated activation of NF‐kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat‐mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV‐1 Tat suppressed the expression of Mn‐dependent superoxide dismutase (Mn‐SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn‐SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn‐SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat1‐72), known to transactivate the HIV‐1 long terminal repeat (LTR), no longer affected Mn‐SOD expression, the cellular redox state or TNF‐mediated cytotoxicity. Thus, our experiments demonstrate that the C‐terminal region of HIV‐1 Tat is required to suppress Mn‐SOD expression and to induce pro‐oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-2 gene expression and NF-kappa B activation through CD28 requires reactive oxygen production by 5-lipoxygenase.

Activation of the CD28 surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of interleukin‐2 (IL‐2) and cell proliferation. In primary T lymphocytes we show that CD28 ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for CD28‐mediated activation of the NF‐kappa B/CD28‐responsive complex and IL‐2 expression. Delineation of the CD28 signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of phospholipase A2 and 5‐lipoxygenase. Our data suggest that lipoxygenase metabolites activate ROI formation which then induce IL‐2 expression via NF‐kappa B activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the CD28 costimulatory pathway.

P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death

Myeloic cells express a peculiar surface receptor for extracellular ATP, called the P2Z/P2X7 purinoreceptor, which is involved in cell death signalling. Here, we investigated the role of caspases, a family of proteases implicated in apoptosis and the cytokine secretion. We observed that extracellular ATP induced the activation of multiple caspases including caspase‐1, ‐3 and ‐8, and subsequent cleavage of the caspase substrates PARP and lamin B. Using caspase inhibitors, it was found that caspases were specifically involved in ATP‐induced apoptotic damage such as chromatin condensation and DNA fragmentation. In contrast, inhibition of caspases only marginally affected necrotic alterations and cell death proceeded normally whether or not nuclear damage was blocked. Our results therefore suggest that the activation of caspases by the P2Z receptor is required for apoptotic but not necrotic alterations of ATP‐induced cell death.

Cancer stem cell markers in common cancers – therapeutic implications

Rapid advances in the cancer stem cell (CSC) field have provided cause for optimism for the development of more reliable cancer therapies in the future. Strategies aimed at efficient targeting of CSCs are becoming important for monitoring the progress of cancer therapy and for evaluating new therapeutic approaches. Here, we characterize and compare the specific markers that have been found to be present on stem cells, cancer cells and CSCs in selected tissues (colon, breast, liver, pancreas and prostate). We then discuss future directions of this intriguing new research field in the context of new diagnostic and therapeutic opportunities.

Redox signalling by transcription factors NF-κB and AP-1 in lymphocytes

When molecular oxygen accumulated in our atmosphere more than 2 billion years ago, aerobic organisms adapted using using oxygen as an electron acceptor in respiration. Although respiration is, by far, one of the most efficient ways of generating energy in biological systems, it has one harmful side-effect: incomplete reduction of dioxygen results in the formation of ROIs. § These include the superoxide anion (02 .-), hydrogen peroxide (H20 2), and the hydroxyl radical (OH·). Superoxide anions originate from a single electron transfer on dioxygen [I]. This ROI has a short diffusion radius inside the cell, but it is eventually converted by SODs into H20 2, the most stable and diffusible ROI. Transition metals, such as iron or copper ions, convert O2.and H20 2 to an extremely reactive hydroxyl radical via the Fenton or Haber/Weiss reaction. While low amounts of ROIs can be considered as physiological side-products of various electron transfer reactions and are tolerated by the cell, increased levels of ROIs, referred to as oxidative stress, are linked to pathophysiological phenomena. Many adverse conditions, including ionizing and UV irradiation, heat shock, viral and bacterial infections, as well as environmental pollutants cause oxidative stress. In principle, ROIs can react with all classes of biological macromolecules, resulting in lipid peroxidation, inactivation of proteins, and strand breakage in nucleic acids [I]. During evolution, cells of the immune system, in particular, have learned to utilize ROIs in two ways: first, as chemical weapons and, second, as signalling molecules triggering a variety of biological processes, such as gene expression and proliferation control. How macrophages and neutrophils produce high amounts of ROIs as a first line of defense against invading microorganisms has been reviewed extensively [2, 3]. However, the roles of ROIs as signalling molecules are just beginning to be discovered. On the one hand, immune cells but also

S100A8/A9 at low concentration promotes tumor cell growth via RAGE ligation and MAP kinase-dependent pathway

The complex formed by two members of the S100 calcium‐binding protein family, S100A8/A9, exerts apoptosis‐inducing activity against various cells, especially tumor cells. Here, we present evidence that S100A8/A9 also has cell growth‐promoting activity at low concentrations. Receptor of advanced glycation end product (RAGE) gene silencing and cotreatment with a RAGE‐specific blocking antibody revealed that this activity was mediated via RAGE ligation. To investigate the signaling pathways, MAPK phosphorylation and NF‐κB activation were characterized in S100A8/A9‐treated cells. S100A8/A9 caused a significant increase in p38 MAPK and p44/42 kinase phosphorylation, and the status of stress‐activated protein kinase/JNK phosphorylation remained unchanged. Treatment of cells with S100A8/A9 also enhanced NF‐κB activation. RAGE small interfering RNA pretreatment abrogated the S100A8/A9‐induced NF‐κB activation. Our data indicate that S100A8/A9‐promoted cell growth occurs through RAGE signaling and activation of NF‐κB.

Caspases: more than just killers?

Proteases of the caspase family constitute the central executioners of apoptosis. Several recent observations suggest that caspases and apoptosis-regulatory molecules exert important functions beyond that of cell death, including the control of T-cell proliferation and cell-cycle progression. Here, Los and colleagues propose a model that directly connects cell suicide mechanisms to the regulation of cell-cycle progression.

Chemosensitivity of solid tumor cells in vitro is related to activation of the CD95 system

We have identified the CD95 system as a key mediator of chemotherapy‐induced apoptosis in leukemia and neuroblastoma cells. Here, we report that sensitivity of various solid tumor cell lines for drug‐induced cell death corresponds to activation of the CD95 system. Upon drug treatment, strong induction of CD95 ligand (CD95‐L) and caspase activity were found in chemosensitive tumor cells (Hodgkin, Ewings sarcoma, colon carcinoma and small cell lung carcinoma) but not in tumor cells which responded poorly to drug treatment (breast carcinoma and renal cell carcinoma). Blockade of CD95 using F(ab′)2 anti‐CD95 antibody fragments markedly reduced drug‐induced apoptosis, suggesting that drug‐triggered apoptosis depended on CD95‐L/receptor interaction. Moreover, drug treatment induced CD95 expression, thereby increasing sensitivity for CD95‐induced apoptosis. Drug‐induced apoptosis critically depended on activation of caspases (ICE/Ced‐3‐like proteases) since the broad‐spectrum inhibitor of caspases zVAD‐fmk strongly reduced drug‐mediated apoptosis. The prototype substrate of caspases, poly(ADP‐ribose) polymerase, was cleaved upon drug treatment, suggesting that CD95‐L triggered autocrine/paracrine death via activation of caspases. Our data suggest that chemosensitivity of solid tumor cells depends on intact apoptosis pathways involving activation of the CD95 system and processing of caspases. Our findings may have important implications for new treatment approaches to increase sensitivity and to overcome resistance of solid tumors. Int. J. Cancer 76:105–114, 1998.© 1998 Wiley‐Liss, Inc.