Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo

Primary cultures of rat type II alveolar epithelial cells (AECs) or human AEC-derived A549 cells, when exposed to bleomycin (Bleo), exhibited concentration-dependent apoptosis detected by altered nuclear morphology, fragmentation of DNA, activation of caspase-3, and net cell loss over time. In both cell culture models, exposure to Bleo caused time-dependent increases in angiotensinogen (ANGEN) mRNA. Antisense oligonucleotides against ANGEN mRNA inhibited Bleo-induced apoptosis of rat AEC or A549 cells by 83 and 84%, respectively (P < 0.01 and P < 0.05), and prevented Bleo-induced net cell loss. Apoptosis of rat AECs or A549 cells in response to Bleo was inhibited 91% by the ANG-converting enzyme inhibitor captopril or 82%, respectively, by neutralizing antibodies specific for ANG II (both P < 0.01). Antagonists of ANG receptor AT(1) (losartan, L-158809, or saralasin), but not an AT(2)-selective blocker (PD-123319), inhibited Bleo-induced apoptosis of either rat AECs (79%, P < 0.01) or A549 cells (83%, P < 0.01) and also reduced the activity of caspase-3 by 52% (P < 0.05). These data indicate that Bleo, like Fas(L) or TNF-alpha, induces transactivation of ANG synthesis de novo that is required for AEC apoptosis. They also support the theory that ANG system antagonists have potential for the blockade of AEC apoptosis in situ.     

Angiotensin receptor subtype AT(1) mediates alveolar epithelial cell apoptosis in response to ANG II

Previous work from this laboratory demonstrated induction of apoptosis in lung alveolar epithelial cells (AEC) by purified angiotensin II (ANG II) and expression of mRNAs for both ANG II receptor subtypes AT(1) and AT(2) (Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 276: L885-L889, 1999.). The present study was designed to determine the ANG II receptor subtype mediating AEC apoptosis in response to ANG II. Apoptosis was induced with purified ANG II applied to the human lung AEC-derived carcinoma cell line A549 or to primary AEC isolated from Wistar rats. In both cell types, the AT(1)-selective receptor antagonists L-158809 or losartan inhibited ANG II-induced apoptosis by 90% at concentrations of 10(-8) M and 10(-7) M, respectively. The inhibition was concentration dependent with IC(50) of 10(-12) M and 10(-11) M on the primary rat AEC. In contrast, the AT(2)-selective antagonists PD-123319 or PD-126055 could not block ANG II-induced apoptosis in either cell type. In primary rat AEC, apoptosis in response to ANG II was blunted in a dose-dependent manner by the protein kinase C inhibitor chelerythrine but not by the tyrosine phosphatase inhibitor sodium orthovanadate. Together, these data indicate that AEC apoptosis in response to ANG II is mediated by receptor subtype AT(1), despite the expression of mRNAs for both AT(1) and AT(2).     

Abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor

Angiotensin-converting enzyme is involved in apoptosis of alveolar epithelial cells (Wang R, Zagariya A, Ang E, Ibarra-Sunga O, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 277: L1245-L1250, 1999). This study tested the ability of the angiotensin-converting enzyme inhibitor captopril or the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD-fmk) to block alveolar epithelial cell apoptosis and lung fibrosis in vivo in response to bleomycin (Bleo). Male Wistar rats received 8 U/kg of Bleo (bleomycin sulfate) or vehicle intratracheally. Subgroups of Bleo-treated rats received captopril, ZVAD-fmk, or vehicle alone. Lung collagen was assessed by picrosirius red or hydroxyproline assay at 1, 7, and 14 days post-Bleo, and apoptosis was detected by in situ end labeling (ISEL). Bleo increased alveolar septal and peribronchial collagen by 100 and 133%, respectively (both P < 0.01), by day 14 but not earlier. In contrast, ISEL was increased in alveolar and airway cells at all time points. Captopril or ZVAD-fmk inhibited collagen accumulation by 91 and 85%, respectively (P < 0. 01). Both agents also inhibited ISEL in alveoli by 99 and 81% and in airways by 67 and 63%, respectively. These data suggest that the efficacy of captopril to inhibit experimental lung fibrogenesis is related to inhibition of apoptosis. They also demonstrate the antifibrotic potential of a caspase inhibitor.     

Norepinephrine induces alveolar epithelial apoptosis mediated by alpha-, beta-, and angiotensin receptor activation

Norepinephrine (NE) induces apoptosis in cardiac myocytes, and autocrine production of angiotensin (ANG) II is required for apoptosis of alveolar epithelial cells (AECs) (Wang R, Zagariya A, Ang E, Ibarra-Sunga O, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 277: L1245--L1250, 1999; Wang R, Alam G, Zagariya A, Gidea C, Pinillos H, Lalude O, Choudhary G, and Uhal BD. J Cell Physiol 185: 253--259, 2000). On this basis, we hypothesized that NE might induce apoptosis of AECs in a manner inhibitable by ANG system antagonists. Purified NE induced apoptosis in the human A549 AEC-derived cell line or in primary cultures of rat AECs, with EC(50) values of 200 and 20 nM, respectively. Neither the alpha-agonist phenylephrine nor the beta-agonist isoproterenol could mimic NE when tested alone but when applied together could induce apoptosis with potency equal to NE. Apoptosis and net cell loss (47--59% in 40 h) in response to NE was completely abrogated by the ANG-converting enzyme inhibitor lisinopril or the ANG II receptor antagonist saralasin, each at concentrations capable of blocking Fas- or tumor necrosis factor-alpha-induced apoptosis. These data suggest that NE induces apoptosis of human and rat AECs through a mechanism involving the combination of alpha- and beta-adrenoceptor activation followed by autocrine generation of ANG II.     

Regulation of alveolar epithelial cell survival by the ACE-2/angiotensin 1-7/Mas axis

Earlier work from this laboratory demonstrated that apoptosis of alveolar epithelial cells (AECs) requires autocrine generation of angiotensin (ANG) II. More recent studies showed that angiotensin converting enzyme-2 (ACE-2), which degrades ANGII to form ANG1-7, is protective but severely downregulated in human and experimental lung fibrosis. Here it was theorized that ACE-2 and its product ANG1-7 might therefore regulate AEC apoptosis. To evaluate this hypothesis, the AEC cell line MLE-12 and primary cultures of rat AECs were exposed to the profibrotic apoptosis inducers ANGII or bleomycin (Bleo). Markers of apoptosis (caspase-9 or -3 activation and nuclear fragmentation), steady-state ANGII and ANG1-7, and JNK phosphorylation were measured thereafter. In the absence of Bleo, inhibition of ACE-2 by small interfering RNA or by a competitive inhibitor (DX600 peptide) caused a reciprocal increase in autocrine ANGII and corresponding decrease in ANG1-7 in cell culture media (both P < 0.05) and, moreover, induced AEC apoptosis. At baseline (without inhibitor), ANG1-7 in culture media was 10-fold higher than ANGII (P < 0.01). Addition of purified ANGII or bleomycin-induced caspase activation, nuclear fragmentation, and JNK phosphorylation in cultured AECs. However, preincubation with ANG1-7 (0.1 μM) prevented JNK phosphorylation and apoptosis. Moreover, pretreatment with A779, a specific blocker of the ANG1-7 receptor mas, prevented ANG1-7 blockade of JNK phosphorylation, caspase activation, and nuclear fragmentation. These data demonstrate that ACE-2 regulates AEC survival by balancing the proapoptotic ANGII and its antiapoptotic degradation product ANG1-7. They also suggest that ANG1-7 inhibits AEC apoptosis through the ANG1-7 receptor mas.     

Amiodarone induces apoptosis of human and rat alveolar epithelial cells in vitro

The antiarrhythmic amiodarone (AM) and its metabolite desethylamiodarone (Des) are known to cause AM-induced pulmonary toxicity, but the mechanisms underlying this disorder remain unclear. We hypothesized that AM might cause AM-induced pulmonary toxicity in part through the induction of apoptosis or necrosis in alveolar epithelial cells (AECs). Two models of type II pneumocytes, the human AEC-derived A549 cell line and primary AECs isolated from adult Wistar rats, were incubated with AM or Des for 20 h. Apoptotic cells were determined by morphological assessment of nuclear fragmentation with propidium iodide on ethanol-fixed cells. Necrotic cells were quantitated by loss of dye exclusion. Both AM and Des caused dose-dependent necrosis starting at 2.5 and 0.1 microg/ml, respectively, in primary rat AECs and at 10 and 5 microg/ml in subconfluent A549 cells (P < 0.05 and P < 0.01, respectively). AM and Des also induced dose-dependent apoptosis beginning at 2.5 microg/ml in the primary AECs (P < 0.05 for both compounds) and at 10 and 5 microg/ml, respectively, in the A549 cell line (P < 0.01). The two compounds also caused significant net cell loss (up to 80% over 20 h of incubation) by either cell type at drug concentrations near or below the therapeutic serum concentration for AM. The cell loss was not due to detachment but was blocked by the broad-spectrum caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone. Furthermore, the angiotensin-converting enzyme inhibitor captopril (500 ng/ml) and the angiotensin-receptor antagonist saralasin (50 microg/ml) significantly inhibited both the induction of apoptosis and net cell loss in response to AM. These results are consistent with recent work from this laboratory demonstrating potent inhibition of apoptosis in human AECs by captopril (Uhal BD, Gidea C, Bargout R, Bifero A, Ibarra-Sunga O, Papp M, Flynn K, and Filippatos G. Am J Physiol Lung Cell Mol Physiol 275: L1013-L1017, 1998). They also suggested that the accumulation of AM and/or its primary metabolite Des in lung tissue may induce cytotoxicity of AECs that might be inhibitable by angiotensin-converting enzyme inhibitors or other antagonists of the renin-angiotensin system.     

Cross-talk between the pathways leading to the induction of apoptosis and the secretion of tumor necrosis factor-alpha in ricin-treated RAW 264.7 cells

Ricin induced apoptotic nuclear morphological changes in mouse macrophage cell line RAW264.7 cells at concentrations sufficient to cause severe protein synthesis inhibition. Ricin also induced the release of tumor necrosis factor-alpha (TNF-alpha) from this cell line in a dose-dependent manner but the profile was bell-shaped. However, the isolated galactose-specific ricin B-chain had no such effects. These results suggest that the receptor-binding of ricin through the B-chain is not enough, and subsequent attack on the intracellular target, i.e., the 28S ribosomal RNA (rRNA), by the A-chain of internalized ricin is required for the effects of ricin. Z-D-CH2-DCB, a caspase family inhibitor, showed potent inhibition of the release of TNF-alpha from RAW264.7 cells as well as blockage of the induction of apoptosis by ricin. Furthermore, SB202190, a specific P38 mitogen-activated protein (MAP) kinase inhibitor that strongly inhibits the release of TNF-alpha, also showed significant inhibition of ricin-induced apoptosis. These results suggest that there may be cross-talk between the pathways leading to the release of TNF-alpha and apoptosis. Time course analysis revealed that the activation of p38 MAP kinase started prior to the induction of TNF-alpha release and apoptosis. Since the activation of p38 MAP kinase in ricin-treated RAW264.7 cells was not prevented by Z-D-CH2-DCB, the activation of p38 MAP kinase may occur upstream of the caspase cascade. Among the other protein synthesis inhibitors examined, modeccin and anisomycin, which can trigger a ribotoxic stress response similar to ricin, induced the release of TNF-alpha, but emetine and cycloheximide did not. These results suggest that the specific attack on the 28S ribosomal RNA and the resulting ribotoxic stress response may trigger the multiple signal transduction pathways through the activation of p38 MAP kinase, which in turn leads to TNF-alpha release and apoptosis.     

Apoptosis induced by TGF-beta 1 in Burkitt's lymphoma cells is caspase 8 dependent but is death receptor independent

TGF-beta is a potent inducer of apoptosis in many Burkitt's lymphoma (BL) cell lines. In this study, we characterize this apoptotic process in the EBV-negative BL41 cell line. Induction of apoptosis was detected as early as 8 h after TGF-beta treatment, as assayed by TUNEL and poly(ADP-ribose) polymerase cleavage. FACS analysis demonstrates that this proceeds predominately from the G1, but also from the G2/M phases of the cell cycle. We observed no early detectable changes in the steady-state levels of Bcl-2 and several of its family members after TGF-beta treatment. We detected cleavage of caspases 2, 3, 7, 8, and 9 into their active subunits. Consistent with the involvement of these enzymes in TGF-beta-mediated apoptosis, the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(Ome)-flouromethylketone (ZVAD-fmk) blocked TGF-beta-induced apoptosis and revealed a G1 arrest in treated cells. Use of specific caspase inhibitors revealed that the induction of apoptosis is caspase 8 dependent, but caspase 3 independent. Activation of caspase 8 has been shown to be a critical event in death receptor-mediated apoptosis. However, TGF-beta treatment of BL41 cells was found not to affect the cell surface expression of Fas, TNF-R1, DR3, DR4, or DR5, or the steady-state expression levels of Fas ligand, TNF-R1, DR3, DR4, and DR5. Furthermore, blocking experiments indicated that TGF-beta-mediated apoptosis is not dependent on Fas ligand, TNF-alpha, tumor necrosis-like apoptosis-inducing ligand, or TNF-like weak inducer of apoptosis signaling. Therefore, it appears that TGF-beta induces apoptosis in BL cell lines via caspase 8 in a death receptor-independent fashion.     

Essential role for cathepsin D in bleomycin-induced apoptosis of alveolar epithelial cells

Our earlier studies showed that bleomycin-induced apoptosis of type II alveolar epithelial cells (AECs) requires the autocrine synthesis and proteolytic processing of angiotensinogen into ANG II and that inhibitors of ANG-converting enzyme (ACEis) block bleomycin-induced apoptosis (Li X, Zhang H, Soledad-Conrad V, Zhuang J, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 284: L501-L507, 2003). Given the documented role of cathepsin D (CatD) in apoptosis of other cell types, we hypothesized that CatD might be the AEC enzyme responsible for the conversion of angiotensinogen into ANG I, the substrate for ACE. Primary cultures of rat type II AECs challenged with bleomycin in vitro showed upregulation and secretion of CatD enzymatic activity and immunoreactive protein but no increases in CatD mRNA. The aspartyl protease inhibitor pepstatin A, which completely blocked CatD enzymatic activity, inhibited bleomycin-induced nuclear fragmentation by 76% and reduced bleomycin-induced caspase-3 activation by 47%. Antisense oligonucleotides against CatD mRNA reduced CatD-immunoreactive protein and inhibited bleomycin-induced nuclear fragmentation by 48%. A purified fragment of angiotensinogen (F1-14) containing the CatD and ACE cleavage sites, when applied to unchallenged AEC in vitro, yielded mature ANG II peptide and induced apoptosis. The apoptosis induced by F1-14 was inhibited 96% by pepstatin A and 77% by neutralizing antibodies specific for CatD (both P < 0.001). These data indicate a critical role for CatD in bleomycin-induced apoptosis of cultured AEC and suggest that the role(s) of CatD in AEC apoptosis include the conversion of newly synthesized angiotensinogen to ANG II.     

FGF-10 prevents mechanical stretch-induced alveolar epithelial cell DNA damage via MAPK activation

Cyclic stretch of alveolar epithelial cells (AEC) can alter normal lung barrier function. Fibroblast growth factor-10 (FGF-10), an alveolar type II cell mitogen that is critical for lung development, may have a role in promoting AEC repair. We studied whether cyclic stretch induces AEC DNA damage and whether FGF-10 would be protective. Cyclic stretch (30 min of 30% strain amplitude and 30 cycles/min) caused AEC DNA strand break formation, as assessed by alkaline unwinding technique and DNA nucleosomal fragmentation. Pretreatment of AEC with FGF-10 (10 ng/ml) blocked stretch-induced DNA strand break formation and DNA fragmentation. FGF-10 activated AEC mitogen-activated protein kinase (MAPK), and MAPK inhibitors prevented FGF-10-induced AEC MAPK activation and abolished the protective effects of FGF-10 against stretch-induced DNA damage. In addition, a Grb2-SOS inhibitor (SH(3)b-p peptide), a RAS inhibitor (farnesyl transferase inhibitor 277), and a RAF-1 inhibitor (forskolin) each prevented FGF-10-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in AEC. Moreover, N17-A549 cells that express a RAS dominant/negative protein prevented the FGF-10-induced ERK1/2 phosphorylation and RAS activation in AEC. We conclude that cyclic stretch causes AEC DNA damage and that FGF-10 attenuates these effects by mechanisms involving MAPK activation via the Grb2-SOS/Ras/RAF-1/ERK1/2 pathway.     

Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release

The proteasome is a multiprotein complex that is involved in the intracellular protein degradation in eukaryotes. Here, we show that human malignant glioma cells are susceptible to apoptotic cell death induced by the proteasome inhibitors, MG132 and lactacystin. The execution of the apoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9. Apoptosis is inhibited by ectopic expression of X-linked inhibitor of apoptosis (XIAP) and by coexposure to the broad-spectrum caspase inhibitor, benzoyl-VAD-fluoromethyl ketone (zVAD-fmk), but not by the preferential caspase 8 inhibitor, crm-A. It is interesting that specific morphological alterations induced by proteasome inhibition, such as dilated rough endoplasmic reticulum and the formation of cytoplasmic vacuoles and dense mitochondrial deposits, are unaffected by zVAD-fmk. Apoptosis is also inhibited by ectopic expression of Bcl-2 or by an inhibitor of protein synthesis, cycloheximide. Further, cytochrome c release and disruption of mitochondrial membrane potential are prominent features of apoptosis triggered by proteasome inhibition. Bcl-2 is a stronger inhibitor of cytochrome c release than zVAD-fmk. XIAP and crm-A fail to modulate cytochrome c release. These data place cytochrome c release downstream of Bcl-2 activity but upstream of XIAP- and crm-A-sensitive caspases. The partial inhibition of cytochrome c release by zVAD-fmk indicates a positive feedback loop that may involve cytochrome c release and zVAD-fmk-sensitive caspases. Finally, death ligand/receptor interactions, including the CD95/CD95 ligand system, do not mediate apoptosis induced by proteasome inhibition in human malignant glioma cells.     

An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.     

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.     

Inhibition of arachidonate 5-lipoxygenase triggers prostate cancer cell death through rapid activation of c-Jun N-terminal kinase

Previously, we reported that inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in both androgen-sensitive (LNCaP) and androgen-refractory (PC3) human prostate cancer cells within hours of treatment [Proc. Natl. Acad. Sci. USA 95 (1998) 13182-13187]. Apoptosis was prevented by exogenous 5(S)-HETE, a product of 5-lipoxygenase, indicating a role of this eicosanoid as an essential survival/anti-apoptotic factor for prostate cancer cells. However, nothing was clearly known about details of the underlying molecular mechanisms or events mediating the induction of fulminating apoptosis in these cells. This report documents the fact that inhibition of arachidonate 5-lipoxygenase induces rapid activation of c-Jun N-terminal kinase (JNK) in human prostate cancer cells which is prevented by the 5-lipoxygenase metabolite, 5(S)-HETE. Activation of JNK is unaffected by the cell-permeable tetra-peptide inhibitors of caspase 8 or caspase 3 (IETD-FMK and DEVD-FMK), though these inhibitors effectively blocked apoptosis triggering, suggesting that activation of JNK is independent or upstream of caspase activation. Both 5-lipoxygenase inhibition-induced activation of JNK and induction of apoptosis are prevented by curcumin, an inhibitor of JNK-signaling pathway. Apoptosis is also blocked by SP600125, a specific inhibitor of JNK activity, indicating that JNK activity is required for the induction of apoptosis in these cells. These findings suggest that the metabolites of arachidonate 5-lipoxygenase promote survival of prostate cancer cells involving down-regulation of stress-activated protein kinase.     

The effect of specific caspase inhibitors on TNF-alpha and butyrate-induced apoptosis of intestinal epithelial cells

Tumour necrosis factor-alpha (TNF-alpha)-induced intestinal epithelial cell apoptosis may contribute to mucosal injury in inflammatory bowel disease. Inhibition of TNF-alpha-induced apoptosis, using specific caspase inhibitors could, therefore, be of benefit in the treatment of disease. In vitro, CaCo-2 colonic epithelial cells are refractory to apoptosis induced by TNF-alpha alone; however, TNF-alpha can act synergistically with the short-chain fatty acid (SCFA) and colonic fermentation product, butyrate, to promote apoptosis. TNF-alpha/butyrate-induced apoptosis was characterised by nuclear condensation and fragmentation and caspase-3 activation. Inhibitors of caspase-8 (z-IETD.fmk) and caspase-10 (z-AEVD.fmk) significantly reduced TNF-alpha/butyrate-induced apoptosis, based on nuclear morphology and terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling (TUNEL), although caspase inhibition was associated with a significant increase in cells demonstrating atypical nuclear condensation. Inclusion of atypical cells in calculations of total cell death, still demonstrated that z-IETD.fmk and z-AEVD.fmk (in combination) significantly reduced cell death. Reduction in cell death was associated with maintenance of viable cell number. Transmembrane resistance was also used a measure of the ability of caspase inhibitors to prevent TNF-alpha/butyrate-mediated damage to epithelial monolayers. TNF-alpha/butyrate resulted in a significant fall in transmembrane resistance, which was prevented by pre-treatment with z-IETD.fmk, but not z-AEVD.fmk. In conclusion, synthetic caspase inhibitors can reduce the apoptotic response of CaCo-2 colonic epithelial cells to TNF-alpha/butyrate, improve the maintenance of viable cell numbers and block loss of transmembrane resistance. We hypothesise that caspase inhibition could be a useful therapeutic goal in the treatment of inflammatory bowel conditions, such as ulcerative colitis.     

The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.     

Apoptosis in sulfur mustard treated A549 cell cultures

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.     

TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages

The Bcg/Nramp1 gene controls early resistance and susceptibility of macrophages to mycobacterial infections. We previously reported that Mycobacterium tuberculosis-infected (Mtb) B10R (Bcgr) and B10S (Bcgs) macrophages differentially produce nitric oxide (NO-), leading to macrophage apoptosis. Since TNF-alpha and IL-10 have opposite effects on many macrophage functions, we determined the number of cells producing TNF-alpha and IL-10 in Mtb-infected or purified protein derivative-stimulated B10R and B10S macrophages lines, and Nramp1+/+ and Nramp1-/- peritoneal macrophages and correlated them with Mtb-mediated apoptosis. Mtb infection and purified protein derivative treatment induced more TNF-alpha+Nramp1+/+ and B10R, and more IL-10+Nramp1-/- and B10S cells. Treatment with mannosylated lipoarabinomannan, which rescues macrophages from Mtb-induced apoptosis, augmented the number of IL-10 B10R+ cells. Anti-TNF-alpha inhibited apoptosis, diminished NO- production, p53, and caspase 1 activation and increased Bcl-2 expression. In contrast, anti-IL-10 increased caspase 1 activation, p53 expression, and apoptosis, although there was no increment in NO- production. Murine rTNF-alpha induced apoptosis in noninfected B10R and B10S macrophages that was reversed by murine rIL-10 in a dose-dependent manner with concomitant inhibition of NO- production and caspase 1 activation. NO- and caspase 1 seem to be independently activated in that aminoguanidine did not affect caspase 1 activation and the inhibitor of caspase 1, Tyr-Val-Ala-Asp-acylooxymethylketone, did not block NO- production; however, both treatments inhibited apoptosis. These results show that Mtb activates TNF-alpha- and IL-10-dependent opposite signals in the induction of macrophage apoptosis and suggest that the TNF-alpha-IL-10 ratio is controlled by the Nramp1 background of resistance/susceptibility and may account for the balance between apoptosis and macrophage survival.     

Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma.

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.