The peroxisome-proliferator-activated receptor (PPAR) γ agonist, CDDO, is under investigation for use in various malignancies. The mechanisms by which CDDO induces apoptosis are controversial. We have therefore sought to determine these mechanisms using primary chronic lymphocyte leukemic (CLL) cells and Jurkat cell lines with defined apoptotic abnormalities. In these cells, CDDO induced-apoptosis involved caspase-independent loss in mitochondrial membrane potential followed by caspase processing. The pattern of CDDO-induced caspase processing, defined by use of a caspase inhibitor, strongly suggested that caspase-9 was the apical caspase. Moreover, CDDO induced apoptosis in caspase-8 and FADD-deficient but not in Bcl-xL overexpressing Jurkat cells. In CLL cells, CDDO induced an early release of mitochondrial cytochrome c and Smac that preceded apoptosis. Thus, in both cell types, CDDO induced apoptosis primarily by the intrinsic pathway with caspase-9 as the apical caspase. This has important implications in the design of novel agents for the treatment of CLL and other malignancies.

New agents are required for the treatment of chronic lymphocytic leukaemia (CLL). We show here that a protein kinase C inhibitor, bisindolylmaleimide IX, is a potent inducer of apoptosis in CLL cells, and investigate the mechanisms by which this is induced. Bisindolylmaleimide IX induced a conformational change and subcellular redistribution of Bax from the cytosol to the mitochondria, resulting in the release of the proapoptotic mediators cytochrome c, Smac and Omi/HtrA2 from the mitochondrial inner membrane space. This was followed by the activation of caspase-9 as the apical caspase and subsequent activation of effector caspases. CLL cells undergoing apoptosis showed a rapid caspase-mediated cleavage of Mcl-1, an antiapoptotic member of the Bcl-2 family implicated in CLL survival and poor prognosis. This cleavage was mediated primarily by caspase-3. Cleavage of Mcl-1 may provide a feed-forward amplification loop, resulting in the rapid induction of apoptosis. Bisindolylmaleimide IX or a related derivative may be of clinical use in the treatment of CLL.

Proteasome inhibitors, including lactacystin and MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), potently induce apoptosis in leukaemic B cells from patients with B cell chronic lymphocytic leukaemia (B-CLL). This pro-apoptotic effect occurs in cells from patients at all stages of the disease, including those resistant to conventional chemotherapy, suggesting that proteasome inhibitors may be useful for treatment of B-CLL. Following initial inhibition of proteasomal activity, these agents induce mitochondrial cytochrome c release and caspase-dependent apoptosis, involving cleavage/activation of caspases -2, -3, -7, -8 and -9. Pre-treatment with the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe)fluoromethyl ketone (Z-VAD.fmk), did not prevent the release of cytochrome c or partial processing of caspase-9 but prevented activation of effector caspases and the induction of apoptosis. These results suggest that the release of cytochrome c is caspase independent and that caspase-9 is the initiator caspase in proteasome inhibitor-induced apoptosis of B-CLL cells. Activation of B-CLL lysates with dATP results in the formation of an 700 kDa caspase-activating apoptosome complex containing Apaf-1. We describe for the first time the formation of a similar 700 kDa caspase-activating apoptosome complex in B-CLL cells induced to undergo apoptosis by proteasome inhibitors.

Goniothalamin, a plant styrylpyrone derivative isolated from Goniothalamus andersonii, induced apoptosis in Jurkat T-cells as assessed by the externalisation of phosphatidylserine. Immunoblotting showed processing of caspases-3 and -7 with the appearance of their catalytically active large subunits of 17 and 19 kDa, respectively. Activation of these caspases was further evidenced by detection of poly(ADP-ribose) polymerase cleavage (PARP). Pre-treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) blocked apoptosis and the resultant cleavage of these caspases and PARP. Our results demonstrate that activation of at least two effector caspases is a key feature of goniothalamin-induced apoptosis in Jurkat T-cells.

Apoptosis is a programmed form of cell death characterized by biochemical and morphological changes affecting the nucleus, cytoplasm, and plasma membrane. These changes in various cellular compartments are widely regarded as mechanistically linked events in a single "program" in which activation of caspases and proteolysis of intracellular substrates represent a final common pathway leading to cell death. To date there has been very limited exploration of the linkage of this program to the plasma membrane changes, which bring about swift recognition, uptake, and safe degradation of apoptotic cells by phagocytes. Using the mitochondrial inhibitors antimycin A and oligomycin in human monocytic THP.1 cells triggered into apoptosis, we report the uncoupling of plasma membrane changes from other features of apoptosis. These inhibitors blocked increased plasma membrane permeability, externalization of phosphatidylserine, and recognition by two classes of phagocytes but not activation of caspase-3, cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. Externalization of phosphatidylserine in apoptotic human leukemic U937 cells was also dissociated from caspase activation. Thus changes governing safe clearance of apoptotic cells may be regulated by an independent pathway to those bringing about caspase activation. This finding could have important consequences for attempts to manipulate cell death for therapeutic gain in vivo.

Dexamethasone and etoposide both induce apoptosis in immature rat thymocytes. We investigated the dependence of apoptosis on the phase of the cell cycle after incubation with these drugs. Cell cycle progression was followed by a combination of pulse labelling with 5-bromo-2'-deoxyuridine (BrdU), labelling fixed cells with an anti-BrdU antibody and flow cytometry. Dexamethasone had little effect on the cell cycle progression of proliferating thymocytes, while etoposide caused cell cycle arrest. Normal and apoptotic thymocytes were separated by centrifugation on discontinuous Percoll gradients into four fractions (F1-F4). It was found that both dexamethasone and etoposide induced apoptosis in cells in G0/G1 and G2/M of the cell cycle, whereas only etoposide induced apoptosis of cells in S phase. These results demonstrated that dexamethasone induced apoptosis in quiescent cells while only etoposide could induce apoptosis in cells from the proliferative compartment. Following treatment of thymocytes with etoposide, some of the proliferating thymocytes (F1) were converted to cells with intermediate size and density (F3). We have recently identified these cells as a population of preapoptotic thymocytes, at an early stage of apoptosis. These cells then further progressed to fully apoptotic cells (F4). These data support the hypothesis that normal thymocytes (F1) became apoptotic (F4) via an intermediate population (F3).

A time- and dose-dependent thymic atrophy was observed in young male Fischer 344 rats dosed intraperitoneally with etoposide (10, 30, or 100 mg/kg). Histopathological examination of the thymus revealed that the pattern of cell death in the majority of thymocytes had a characteristic apoptotic morphology typified by nuclear condensation. This observation was supported by the formation of internucleosomal fragments of DNA in thymocytes isolated from animals dosed with etoposide. Administration of the protein synthesis inhibitor, cycloheximide (1.5 mg/kg, ip), 1 hr prior to etoposide inhibited the induction of apoptosis in thymocytes, assessed by both biochemical and histological criteria. Flow cytometric analysis of thymocytes from animals dosed with etoposide in vivo revealed the formation of both apoptotic cells and apoptotic bodies in contrast to previous in vitro studies which showed the formation of only apoptotic cells. Our data indicate that the induction of apoptosis in thymocytes is a major mechanism involved in etoposide-induced thymic atrophy.

Etoposide, a DNA topoisomerase II inhibitor, caused a concentration-dependent induction of apoptosis in immature thymocytes. Using a flow cytometric method to separate and quantify normal and apoptotic cells, etoposide-induced apoptosis was inhibited by cycloheximide and actinomycin D but not by zinc. Etoposide induced a marked cleavage of DNA into nucleosomal length fragments or multiples thereof, which was completely inhibited if the thymocytes were also incubated in the presence of zinc. Etoposide, alone, induced the classical ultrastructural features of apoptosis, but in the presence of zinc, the morphological pattern was markedly different and dominated by discrete clumps of condensed chromatin abutting the nuclear membrane. These latter changes resemble those described as the earliest changes in apoptosis. These results support the hypothesis that, in the induction of apoptosis, critical alterations in nuclear chromatin occur prior to endonuclease cleavage of DNA into nucleosomal fragments.

Apoptosis is a process in which cells die in a controlled manner and apparently participate in their own demise. It is best characterized morphologically by condensation of chromatin and biochemically by cleavage of chromatin at internucleosomal regions to yield a classical DNA ladder pattern. Apoptosis was induced in thymocytes by exposure to either the glucocorticoid, dexamethasone, or DNA topoisomerase II inhibitor, etoposide. We describe the formation of large m.w. fragments of DNA, 30 to 50 kilobase pairs in length, in a population of these thymocytes at an early stage of apoptosis before internucleosomal cleavage of DNA. These fragments are absent in normal thymocytes and their formation is dependent on protein synthesis. Their appearance is coincident with the commitment of these cells to apoptosis. The formation of these large fragments is associated with the condensation of chromatin, abutting the nuclear membrane, recognized as one of the earliest ultrastructural signs of apoptosis. Subsequent cleavage of these large fragments gives rise to oligonucleosomal fragments and is independent of protein synthesis. We propose that the formation of large fragments of DNA represents a key committed step in apoptosis, and that it is from these fragments that the archetypal DNA ladders associated with apoptosis are derived.

Inhibitors of macromolecular synthesis and topoisomerases induce apoptosis in the human leukaemic cell line, U937. In this study, U937 cells were treated with the RNA synthesis inhibitor, actinomycin D (1 microM), the protein synthesis inhibitors, emetine (1 microM) and cycloheximide (100 microM), the topoisomerase II inhibitor, teniposide (5 microM), or the topoisomerase I inhibitor, camptothecin (1 microM). Apoptotic cell death was assessed both by flow cytometry and agarose gel electrophoresis, and was correlated to the appearance of large (20 to > or = 580 kilobase pairs) DNA fragments, as assessed by field inversion gel electrophoresis. In all cases, the appearance of DNA fragments of 20-50 kilobase pairs accompanied the appearance of an apoptotic population and of internucleosomal cleavage. However, teniposide additionally induced a marked increase in fragmentation to > or = 580 kilobase pairs. The cotreatment of cells with zinc (1 mM) inhibited the formation of all large DNA fragments, internucleosomal cleavage and the appearance of an apoptotic population. We conclude that the generation of large DNA fragments is characteristic of apoptosis induced by various stimuli in U937, as has been found previously in rat thymocytes. However, unlike what occurs in rat thymocytes, zinc treatment does not dissociate the formation of large fragments from conventional markers of apoptosis.

Recent in vitro studies have suggested that activation of apoptosis could account for the profound depletion of cortical thymocytes, which characterizes tributyltin (TBT) immunotoxicity. However, it has also been shown that TBT disrupts macro-molecular synthesis and cellular energetics to an extent that might be expected to interfere with the initiation of apoptosis. The purpose of these studies was to further evaluate the morphological and biochemical characteristics of thymocyte killing by IRT and to relate this to key cellular processes. Ex vivo thymocyte cultures from immature rats were treated with bis(tri-n-butyltin) oxide (TBTO) at concentrations ranging from those which rapidly produced necrosis (5-10 μM), down to cytotoxic but subnecrotic concentrations (0.1-2 μM). In cells exposed to IBTO concentrations that caused a rapid and near maximal inhibition of protein synthesis, it remained possible to demonstrate the stereotypic internucleosomal DNA cleavage and morphological changes indicative of apoptosis. Further confirmation that apoptosis was occurring independently from protein synthesis was provided by the absence of a protective effect following cycloheximide pretreatment. Apoptosis still occurred in TBTO-treated thymocytes although intracellular ATP levels were depressed to 20% or less of control values. Cytoprotective effects were noted with the intracellular Ca2+ chelators BAPTA-AM and Quin-2 AM, and also with zinc. Cell killing by TBTO occurred without overt disturbance of thymocyte cell cycle parameters. These results indicate that thymocyte apoptosis stimulated by TBT exposure occurs independently of a requirement for protein synthesis and does not require fully conserved cellular energetics.

A need exists for an appropriate animal model for the involvement of both hepatitis B virus infection and ingestion of aflatoxins in the etiology of liver cancer. Duck hepatitis B virus-infected ducks, on the basis of hepatoma development in the wild in China, appear to offer this possibility. The duck has been reexamined as a model system, and key metabolic processes have been assayed in comparison with the rat model for hepatocarcinogenesis. Aflatoxin B1 was found to be more actively metabolized by hepatic microsomes isolated from Pekin ducks in vitro to the aflatoxin B1-8,9-epoxide than corresponding fractions from the rat, and in vivo, higher levels of aflatoxin B1-guanine adduct were formed in hepatic DNA than in the livers of the aflatoxin B1-sensitive F344 rat. Repair of this DNA lesion in the duck and the subsequent formation of the ring-opened aflatoxin B1-FAPy adduct paralleled that in the rat. No effect of duck hepatitis B virus infection was found on any of these biochemical processes. The formation of hepatic lesions was also studied, and lesions were compared with those seen in the aflatoxin B1-treated rat. Histological analysis of necropsy specimens from ducks, 20 mo after the ducks received doses of aflatoxin B1 (25 and 50 micrograms/kg body wt), showed almost complete regression of the early acute lesions, with no evidence of neoplasia. Male F344 rats treated with aflatoxin B1 150 micrograms/kg 5 days/wk for 4 wk had extensive bile duct hyperplasia at the end of the treatment period and 100% incidence of hepatocellular carcinoma after 52 wk. The possible basis for the relative sensitivity of ducks and rats to the carcinogenic action of aflatoxin B1 is discussed.

In normal rats just before weaning the majority of hepatocytes are mononucleated diploids, but within days the number of binucleated cells reaches a peak (approximately 50%) before declining again and there is a steady shift of diploid to tetraploid nuclei. When weanling rats were exposed to ethoxyquin (EQ), the conversion of 2N nuclei to 4N and 8N nuclei as measured by flow cytometry was slowed down. The rapid rise in the number of binucleate cells was also delayed, although the long-term effect was an increased number compared with age-matched controls. It appeared that when EQ was present in the diet, significant numbers of diploid hepatocytes undergoing DNA synthesis also underwent mitosis and cytokinesis giving rise to new diploid hepatocytes. However, many hepatocytes from animals maintained on a control diet did not undergo cytokinesis. Thus the slower 'conversion' of 2N to 4N nuclei in treated hepatocytes was due in part to promotion of cytokinesis in diploid cells undergoing DNA synthesis. The ploidy of a cell would be expected to affect gene expression. EQ is a very potent inducer of gamma-glutamyltranspeptidase (GGT), but expression depended on the age of the animals, the length of treatment time and apparently the ploidy status of the liver. In weanling rats treated with EQ for 7 days, >80% of the hepatocytes expressed GGT, while in 42 day old rats similarly treated < 50% were positive for this enzyme. GGT expression was closely correlated with the percentage of 2N nuclei present in hepatocytes, suggesting that it was more easily induced in cells containing these nuclei than in those containing nuclei of higher ploidy. Although butylated hydroxytoluene (BHT), at the same concentration in the diet, had a similar negative effect on weight gain as did EQ, it had no effect on ploidy, nor did it induce GGT to the same extent as EQ.

We have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thymocytes.

Apoptosis and necrosis are two important mechanisms of cell death. Several methods have recently been described for quantifying apoptotic cells by flow cytometry. We report a novel method for the quantification and separation of viable normal and apoptotic cells. We have applied this method both to immature rat thymocytes treated with a variety of agents and to a murine haemopoetic cell line after withdrawal of a growth factor. The cells were incubated with two dyes which give fluorescent complexes when bound to DNA, the bis-benzimidazole, Hoechst 33342, and propidium iodide. Three populations were identified and characterized. On excitation with UV radiation, dead cells fluoresced red due to the uptake of propidium iodide whereas apoptotic cells fluoresced bright blue; normal cells showed low blue, low red fluorescence. In this paper, we demonstrate how this method may be used to help to distinguish between cell death by apoptosis and necrosis.

Apoptosis, a major form of cell death. is characterized by chromatin condensation, a reduction in cell volume and endonuclease cleavage of DNA into oligonucleosomal length fragments. The detection of these fragments by gel electrophoresis, as a DNA ladder, is currently used as the major biochemical index of apoptosis. Here we report that key morphological changes of apoptosis can be dissociated experimentally from the DNA fragmentation produced by endonuclease activity. Internucleosomal cleavage of DNA is thus likely to be a later event in the apoptotic process.

The induction of apoptosis by glucocorticoids in isolated thymocytes has been studied extensively. However, it is not known whether or not the same changes occur after in vivo glucocorticoid treatment. In order to investigate this, we have studied the changes occurring in thymocytes isolated from rats, from 2-24 hr after a dose of dexamethasone (1 mg/kg), which caused 50% thymic atrophy. Thymocytes were separated into four fractions by isopycnic Percoll gradients. A loss of cells occurred within 2-8 hr, primarily in only one of the two major fractions of normal thymocytes. This loss of normal thymocytes coincided with the appearance of small dense cells with characteristic features of apoptosis including condensed chromatin, increased DNA fragmentation, internucleosomal DNA cleavage and a "hypodiploid" peak on flow cytometric analysis. Striking differences occurred in the cellular composition of the different Percoll fractions with time. Initially (up to 4 hr), the pattern of changes occurring in vivo resembled those found in vitro. However, at later times, the complex fate of apoptotic cells in vivo, such as phagocytosis, are not observed in the in vitro studies.

Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.