A cell's agony of choice: how to cross the Styx? : From morphological to molecular approaches to disclose its decision.

The original "apoptosis-necrosis" concept was based on morphology and (patho)physiological conditions of the occurrence of cell death: (1) apoptosis, with nuclear and cytoplasmic condensation/fragmentation prominent, exclusion of autolysis, considered to result from coordinated self-destruction of a cell; (2) necrosis, with cell lysis prominent, caused by violent environmental perturbation leading to collapse of internal homeostasis. This suggestion initiated a controversial discussion within the scientific community and it soon became clear that the "apoptosis-necrosis dichotomy" was not generally applicable. Nowadays, there is sufficient evidence that cells may activate diverse suicide pathways, thereby allowing a flexible response to environmental changes, either physiological or pathological. The present paper commemorates electron microscopic and cytochemical studies on cell death of cultured human mammary carcinoma cells performed by Adi Ellinger, adding a significant contribution to recognize that autophagy can be involved in regulated cell death, thereby challenging the apoptosis-necrosis dichotomy still predominant in the 1990s.

Cell death and autophagy: cytokines, drugs, and nutritional factors.

Cells may use multiple pathways to commit suicide. In certain contexts, dying cells generate large amounts of autophagic vacuoles and clear large proportions of their cytoplasm, before they finally die, as exemplified by the treatment of human mammary carcinoma cells with the anti-estrogen tamoxifen (TAM, < or = 1 microM). Protein analysis during autophagic cell death revealed distinct proteins of the nuclear fraction including GST-pi and some proteasomal subunit constituents to be affected during autophagic cell death. Depending on the functional status of caspase-3, MCF-7 cells may switch between autophagic and apoptotic features of cell death [Fazi, B., Bursch, W., Fimia, G.M., Nardacci R., Piacentini, M., Di Sano, F., Piredda, L., 2008. Fenretinide induces autophagic cell death in caspase-defective breast cancer cells. Autophagy 4(4), 435-441]. Furthermore, the self-destruction of MCF-7 cells was found to be completed by phagocytosis of cell residues [Petrovski, G., Zahuczky, G., Katona, K., Vereb, G., Martinet, W., Nemes, Z., Bursch, W., Fésüs, L., 2007. Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes. Cell Death Diff. 14 (6), 1117-1128]. Autophagy also constitutes a cell's strategy of defense upon cell damage by eliminating damaged bulk proteins/organelles. This biological condition may be exemplified by the treatment of MCF-7 cells with a necrogenic TAM-dose (10 microM), resulting in the lysis of almost all cells within 24h. However, a transient (1h) challenge of MCF-7 cells with the same dose allowed the recovery of cells involving autophagy. Enrichment of chaperones in the insoluble cytoplasmic protein fraction indicated the formation of aggresomes, a potential trigger for autophagy. In a further experimental model HL60 cells were treated with TAM, causing dose-dependent distinct responses: 1-5 microM TAM, autophagy predominant; 7-9 microM, apoptosis predominant; 15 microM, necrosis. These phenomena might be attributed to the degree of cell damage caused by tamoxifen, either by generating ROS, increasing membrane fluidity or forming DNA-adducts. Finally, autophagy constitutes a cell's major adaptive (survival) strategy in response to metabolic challenges such as glucose or amino acid deprivation, or starvation in general. Notably, the role of autophagy appears not to be restricted to nutrient recycling in order to maintain energy supply of cells and to adapt cell(organ) size to given physiological needs. For instance, using a newly established hepatoma cell line HCC-1.2, amino acid and glucose deprivation revealed a pro-apoptotic activity, additive to TGF-beta1. The pro-apoptotic action of glucose deprivation was antagonized by 2-deoxyglucose, possibly by stabilizing the mitochondrial membrane involving the action of hexokinase II. These observations suggest that signaling cascades steering autophagy appear to provide links to those regulating cell number. Taken together, our data exemplify that a given cell may flexibly respond to type and degree of (micro)environmental changes or cell death stimuli; a cell's response may shift gradually from the elimination of damaged proteins by autophagy and the recovery to autophagic or apoptotic pathways of cell death, the failure of which eventually may result in necrosis.

The activin axis in liver biology and disease.

Activins are a closely related subgroup within the TGFbeta superfamily of growth and differentiation factors. They consist of two disulfide-linked beta subunits. Four mammalian activin beta subunits termed beta(A), beta(B), beta(C), and beta(E), respectively, have been identified. Activin A, the homodimer of two beta(A) subunits, has important regulatory functions in reproductive biology, embryonic development, inflammation, and tissue repair. Several intra- and extracellular antagonists, including the activin-binding proteins follistatin and follistatin-related protein, serve to fine-tune activin A activity. In the liver there is compelling evidence that activin A is involved in the regulation of cell number by inhibition of hepatocyte replication and induction of apoptosis. In addition, activin A stimulates extracellular matrix production in hepatic stellate cells and tubulogenesis of sinusoidal endothelial cells, and thus contributes to restoration of tissue architecture during liver regeneration. Accumulating evidence from animal models and from patient data suggests that deregulation of activin A signaling contributes to pathologic conditions such as hepatic inflammation and fibrosis, acute liver failure, and development of liver cancer. Increased production of activin A was suggested to be a contributing factor to impaired hepatocyte regeneration in acute liver failure and to overproduction of extracellular matrix in liver fibrosis. Recent evidence suggests that escape of (pre)neoplastic hepatocytes from growth control by activin A through overexpression of follistatin and reduced activin production contributes to hepatocarcinogenesis. The role of the activin subunits beta(C) and beta(E), which are both highly expressed in hepatocytes, is still quite incompletely understood. Down-regulation in liver tumors and a growth inhibitory function similar to that of beta(A) has been shown for beta(E). Contradictory results with regard to cell proliferation have been reported for beta(C). The profound involvement of the activin axis in liver biology and in the pathogenesis of severe hepatic diseases suggests activin as potential target for therapeutic interventions.

No increase of apoptosis in regressing mouse liver after withdrawal of growth stimuli or food restriction.

In short-term in vivo experiments, liver growth and regression in mice with high (C3H/He), intermediate (B6C3F1) or low (C57BL/6J) susceptibility to hepatocarcinogenesis was compared. Liver growth was induced by dietary administration of phenobarbital (PB; 750 ppm) or nafenopin (NAF; 500 ppm). PB or NAF treatment for 7 days produced moderate increases of liver DNA (15% or 25-28%, respectively) along with pronounced hypertrophy. Liver growth was strongest in C3H/He mice. Cessation of PB or NAF treatment led to a rapid regression of liver hypertrophy. However, the enhanced hepatic DNA content persisted for at least 2 weeks in all mouse strains. Apoptosis was not increased at any time after cessation of treatment in all strains. Food restriction to 60% of the ad libitum intake did not amplify either regression of liver hyperplasia or the occurrence of apoptosis. No strain difference in the occurrence of apoptosis was detected. Mouse hepatocytes in liver regressing after mitogen withdrawal do not enter apoptosis as readily as rat hepatocytes.

Apoptosis in stages of mouse hepatocarcinogenesis: failure to counterbalance cell proliferation and to account for strain differences in tumor susceptibility.

C3H/He and B6C3F1 show much higher liver cancer susceptibility than C57BL/6J mice. We studied the hypothesis that this difference might result from failure of apoptosis. Hepatocarcinogenesis was induced by a single dose of N-nitrosodiethylamine (NDEA), followed by phenobarbital (PB) for up to 90 weeks. We observed (1) earlier appearance of putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and carcinoma (HCC) in C3H/He than in C57Bl/6J mice and (2) an increase of hepatocellular DNA synthesis in C3H/He and C57Bl/6J mice, compared to normal liver, via PPF and HCA to HCC. PB enhanced DNA synthesis and growth of PPF, in the C3H/He strain only, and of HCA and HCC of both strains. Apoptoses were rare in unaltered livers as well as in preneoplastic lesions, but tended to increase in HCA and HCC of both strains. PB lowered apoptotic activity in PPF of C3H/He mice, but enhanced it in HCA and HCC of C57Bl/6J mice at late stages. In conclusion, the strain difference in growth rates of PPF and tumors is largely determined by higher rates of cell proliferation in C3H/He mice, with and without promotion by PB. Moreover, in C57Bl/6J mice the promoting effect of PB was restricted to HCA and HCC and was not seen in PPF. Apoptosis was generally low and was not a major cause of the strain difference in tumor susceptibility. In contrast with rat liver, inhibition of apoptosis appears to be a minor determinant of tumor promotion in mice.

Autophagy--a basic mechanism and a potential role for neurodegeneration.

Autophagy constitutes a fundamental survival strategy of cells; its disturbance contributes to the pathogenesis of cancer, liver and immune disease, pathogen infection, myopathies as well as neurodegenerative disorders such as Amyotrophic lateral sclerosis, Parkinson;s, Huntington;s and Alzheimer;s disease. The pathogenesis of neurodegenerative diseases also involves a gradual and progressive loss of neuronal cells. Cells may use different pathways for active self-destruction as reflected by different morphology: while in apoptosis (or "type I") nuclear fragmentation associated with cytoplasmic condensation but preservation of organelles is predominant, autophagic degradation of the cytoplasmic structures preceding nuclear collapse is a characteristic of a second type of programmed cell death (PCD). Linking autophagy to programmed cell death initiated a controversial discussion on how a suggested role of autophagy in cell suicide might meet with its established survival function. To some extent, the diverse morphologies can be associated with distinct biochemical and molecular events [caspase-dependent and -independent death programs, DAP-kinase activity, Ras-expression, induction of autophagy genes, fate of cytoskeleton, among others]. However, there is a broad overlap between cell death pathways. Conceivably, diverse PCD programs emerged during evolution, the conservation of which allows eukaryotic cells a flexible response to physiological or pathological demands.

Induction of apoptosis in mouse liver adenoma and carcinoma in vivo by transforming growth factor-beta1.

PURPOSE: In the liver, transforming growth factor beta-1 (TGF-beta1) constitutes a major negative growth regulating factor involved in the control of cell numbers; failure of this control mechanism has been associated with the development of liver cancer. Since no reports on the in vivo effects of exogenously administered TGF-beta1 on apoptosis in liver tumors have been published yet, we studied hepatocyte sensitivity to the proapoptotic action of TGF-beta1 in stages of chemically induced mouse liver carcinogenesis. METHODS: Mouse liver carcinogenesis was initiated by a single dose of N-nitrosodiethylamine (NDEA, 90 mg/kg b.w., i.p.) to 5-week-old B6C3F1 mice. After 2 weeks, mice received either standard diet or a diet containing phenobarbital (PB, 90 mg/kg b.w) for 85 weeks. Four hours before being killed mice received a single dose of TGF-beta1 (56 microg or 200 microg TGF-beta1/kg of b.w., injected into the tail vein). Quantitative histological analysis of mitosis and apoptosis in normal liver tissue (NL), putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) was performed on H&E-stained liver sections. RESULTS: In NDEA and NDEA + PB-treated mice, NL exhibited a very low incidence of apoptosis and mitosis, which increased in HCA and HCC. In the lesions apoptoses ranged between 0.03 and 0.6%. Two hundred micrograms of TGF-beta1/kg stimulated apoptoses in NL as well as in neoplastic lesions (significant increase in NL, HCA, and HCC); the most pronounced proapoptotic action of TGF-ss1 was observed in lesions of NDEA+PB pretreated mice (about 1.7%). Fifty-six microg TGF-beta1/kg had no detectable effect on apoptosis. CONCLUSION: These observations indicate that during chemically induced liver carcinogenesis in B6C3F1 mice basal rates of apoptoses in adenoma and carcinoma are higher than in normal liver and can be further increased by a proapoptotic cytokine.

2-deprenyl-rheediaxanthone B isolated from Metaxya rostrata induces active cell death in colorectal tumor cells.

Metaxya rostrata C. Presl (Metaxyaceae) is a common tree fern in Central and South America that is used for the treatment of intestinal ulcers and tumours in ethnic medicine. Using a bioactivity-guided strategy 2-deprenyl-rheediaxanthone B (XB) has been isolated as one of the active principles in this plant. XB induced loss of cell viability in colorectal cancer cell lines at IC50 concentrations of 11-23 µM. This was caused by both accumulation of cells in the G2- and S-phase as well as by induction of active cell death in a time and concentration-dependent manner. Cells exposed to XB were incapable of undergoing regular mitosis due to down-regulation of FoxM1 and absence of chromosome condensation. The apoptosis-related proteins Bcl2 and Bclxl were up-regulated so that Caspase 3 was not activated and classical apoptosis was not observed. However, XB triggered damage pathways down-stream of ATR and activated Caspase 2 causing cell death by a mechanism similar to mitotic catastrophe. Our observations are the first to show the cytotoxic activity of 2-deprenyl-rheediaxanthone B and indicate that XB is an interesting new lead compound for cancer therapy that merits further development.

Wnt/beta-catenin signaling activates and determines hepatic zonal expression of glutathione S-transferases in mouse liver.

Glutathione S-transferases (GSTs) play an essential role in the elimination of xenobiotic-derived electrophilic metabolites and also catalyze certain steps in the conversion of endogenous molecules. Their expression is controlled by different transcription factors, such as the antioxidant-activated Nrf2 or the constitutive androstane receptor. Here, we show that the Wnt/beta-catenin pathway is also involved in the transcriptional regulation of GSTs: GSTm2, GSTm3, and GSTm6 are overexpressed in mouse hepatomas with activating Ctnnb1 (encoding beta-catenin) mutations and in transgenic hepatocytes expressing activated beta-catenin. Inversely, GSTm expression is reduced in mice with hepatocyte-specific knock out of Ctnnb1. Activation of beta-catenin-dependent signaling stimulates GSTm expression in vitro. Activation of beta-catenin in mouse hepatoma cells activates GSTm3 promoter-driven reporter activity, independently of beta-catenin/T-cell factor sites, via a retinoid X receptor-binding site. By contrast, GSTm expression is inhibited upon Ras activation in mouse liver tumors and transgenic hepatocytes. Recent studies by different groups have shown that beta-catenin-dependent signaling is involved in the transcriptional control of "perivenous" expression of various cytochrome P450s in mouse liver, whereas Ras signaling was hypothesized to antagonize the perivenous hepatocyte phenotype. In synopsis with our present results, it now appears that the Wnt/beta-catenin pathway functions as a master regulator of the expression of both phase I and phase II drug-metabolizing enzymes in perivenous hepatocytes from mouse liver.

Fenretinide induces autophagic cell death in caspase-defective breast cancer cells.

The elimination of tumor cells by apoptosis is the main mechanism of action of chemotherapeutic drugs. More recently, autophagic cell death has been shown to trigger a nonapoptotic cell death program in cancer cells displaying functional defects of caspases. Fenretinide (FenR), a synthetic derivative of retinoic acid, promotes growth inhibition and induces apoptosis in a wide range of tumor cell types. The present study was designed to evaluate the ability of fenretinide to induce caspase-independent cell death and to this aim we used the human mammary carcinoma cell line MCF-7, lacking functional caspase-3 activity. We demonstrated that in these cells fenretinide is able to trigger an autophagic cell death pathway. In particular we found that fenretinide treatment resulted in the increase in Beclin 1 expression, the conversion of the soluble form of LC3 to the autophagic vesicle-associated form LC3-II and its shift from diffuse to punctate staining and finally the increase in lysosomes/autophagosomes. By contrast, caspase-3 reconstituted MCF-7 cell line showed apoptotic cell death features in response to fenretinide treatment. These data strongly suggest that fenretinide does not invariably elicit an apoptotic response but it is able to induce autophagy when apoptotic pathway is deregulated. The understanding of the molecular mechanisms involved in fenretinide action is important for the future design of therapies employing this retinoid in breast cancer treatment.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

Multiple cell death programs: Charon's lifts to Hades.

Cells use different pathways for active self-destruction as reflected by different morphology: while in apoptosis (or "type I") nuclear fragmentation associated with cytoplasmic condensation but preservation of organelles is predominant, autophagic degradation of cytoplasmic structures preceding nuclear collapse is a characteristic of a second type of programmed cell death (PCD). The diverse morphologies can be attributed--at least to some extent--to distinct biochemical and molecular events (e.g. caspase-dependent and -independent death programs; DAP-kinase activity, Ras-expression). However, apoptosis and autophagic PCD are not mutually exclusive phenomena. Rather, diverse PCD programs emerged during evolution, the conservation of which apparently allows cells a flexible response to environmental changes, either physiological or pathological.

Immunohistochemical detection of activated caspases in apoptotic hepatocytes in rat liver.

In our study we tested the utility of antibodies that specifically recognize the cleaved large (active) subunits of caspase-3 and caspase-9 for immunohistochemical detection of apoptotic hepatocytes in rat liver sections using archival material from cyproterone acetate (CPA)-treated and control rats. CPA blocks apoptosis of hepatocytes and discontinuation of CPA treatment results in a syncronized wave of hepatocyte apoptosis. By comparing liver sections from CPA-treated and control rats with high and low rates of apoptosis we observed a close correlation between the occurrence of cleaved caspase-positive apoptotic figures and H&E-stained apoptotic bodies when evaluated in parallel sections. Caspase-stained figures were either immuno-positive apoptotic bodies or pre-apoptotic hepatocytes showing cytoplasmic and/or nuclear caspase-staining with otherwise normal cellular appearance. In extension of these observations we developed a double-immunohistochemical staining procedure which enables the detection of caspase-3-positive apoptotic hepatocytes within glutathione-S-transferase-P (GST-P)-positive preneoplastic liver foci. By use of this technique, inhibition of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin as detected by counting of H&E-stained apoptotic bodies was found to correlate with a strong reduction of cleaved caspase-positive hepatocytes in GST-P-positive preneoplastic foci. In summary, this study demonstrates that cleaved caspase-positive apoptotic hepatocytes could be reliably identified and quantified both in normal and neoplastically transformed liver tissue.