Destructive Changes in the Neuronal Structure of the FVB/N Mouse Retina.

We applied a series of selective antibodies for labeling the various cell types in the mammalian retina. These were used to identify the progressive loss of neurons in the FVB/N mouse, a model of early onset retinal degeneration produced by a mutation in the pde6b gene. The immunocytochemical studies, together with electroretinogram (ERG) recordings, enabled us to examine the time course of the degenerative changes that extended from the photoreceptors to the ganglion cells at the proximal end of the retina. Our study indicates that photoreceptors in FVB/N undergo a rapid degeneration within three postnatal weeks, and that there is a concomitant loss of retinal neurons in the inner nuclear layer. Although the loss of rods was detected at an earlier age during which time M- and S-opsin molecules were translocated to the cone nuclei; by 6 months all cones had also degenerated. Neuronal remodeling was also seen in the second-order neurons with horizontal cells sprouting processes proximally and dendritic retraction in rod-driven bipolar cells. Interestingly, the morphology of cone-driven bipolar cells were affected less by the disease process. The cellular structure of inner retinal neurons, i.e., ChAT amacrine cells, ganglion cells, and melanopsin-positive ganglion cells did not exhibit any gross changes of cell densities and appeared to be relatively unaffected by the massive photoreceptor degeneration in the distal retina. However, Muller cell processes began to express GFAP at their endfeet at p14, and it climbed progressively to the cell's distal ends by 6 months. Our study indicates that FVB/N mouse provides a useful model with which to assess possible intervention strategies to arrest photoreceptor death in related diseases.

Potassium channel Kv2.1 is regulated through protein phosphatase-1 in response to increases in synaptic activity.

The functional stability of neurons in the face of large variations in both activity and efficacy of synaptic connections suggests that neurons possess intrinsic negative feedback mechanisms to balance and tune excitability. While NMDA receptors have been established to play an important role in glutamate receptor-dependent plasticity through protein dephosphorylation, the effects of synaptic activation on intrinsic excitability are less well characterized. We show that increases in synaptic activity result in dephosphorylation of the potassium channel subunit Kv2.1. This dephosphorylation is induced through NMDA receptors and is executed through protein phosphatase-1 (PP1), an enzyme previously established to play a key role in regulating ligand gated ion channels in synaptic plasticity. Dephosphorylation of Kv2.1 by PP1 in response to synaptic activity results in substantial shifts in the inactivation curve of IK, resulting in a reduction in intrinsic excitability, facilitating negative feedback to neuronal excitability.

Glycinergic feedback enhances synaptic gain in the distal retina.

Glycine input originates with interplexiform cells, a group of neurons situated within the inner retina that transmit signals centrifugally to the distal retina. The effect on visual function of this novel mechanism is largely unknown. Using gramicidin-perforated patch whole cell recordings, intracellular recordings and specific antibody labelling techniques, we examined the effects of the synaptic connections between glycinergic interplexiform cells, photoreceptors and bipolar cells. To confirm that interplexiform cells make centrifugal feedback on bipolar cell dendrites, we recorded the postsynaptic glycine currents from axon-detached bipolar cells while stimulating presynaptic interplexiform cells. The results show that glycinergic interplexiform cells activate bipolar cell dendrites that express the α3 subunit of the glycine receptor, as well as a subclass of unidentified receptors on photoreceptors. By virtue of their synaptic contacts, glycine centrifugal feedback increases glutamate release from photoreceptors and suppresses the uptake of glutamate by the type 2A excitatory amino acid transporter on photoreceptors. The net effect is a significant increase in synaptic gain between photoreceptors and their second-order neurons.

Dopamine modulates the off pathway in light-adapted mouse retina.

DL-2-Amino-4-phosphonobutyric acid (APB) is often used as a tool to block On pathways in studies of interactions between On and Off pathways in retinas. APB is an agonist of mGluR6 receptors and hyperpolarizes the On cone bipolar cells and rod bipolar cells. How APB affects Off responses of retinal ganglion cells (RGCs) in mouse retinas under dark and light adaptation is not clear. The light-evoked excitatory postsynaptic currents (light-evoked EPSCs) from Off and On-Off RGCs cells were recorded using whole-cell patch-clamp recording to assess how APB affects Off responses (light-evoked Off EPSCs) of RGCs in dark- and light-adapted mouse retinas. We found that APB differentially affected Off responses of RGCs in dark- and light-adapted mouse retinas. Under dark adaptation, while the APB-sensitive Off responses were blocked, APB increased the remaining Off responses (mainly from the secondary rod Off pathways) via removal of inhibition from On pathways to Off pathways. Under light adaptation, APB decreased Off responses. Glycinergic and GABAergic antagonists did not prevent the APB-induced reduction of Off responses of RGCs; however, a dopaminergic type 1 receptor (D(1)) blocker (SCH 23390) and a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker (ZD 7288) prevented the APB-induced reduction of Off responses of RGCs under light adaptation. The results indicated afunctional circuit: On cone bipolar cells to Off cone bipolar cells via D(1) receptors and HCN channels.

Hyaluronan Does Not Affect Bupivacaine's Inhibitory Action on Voltage-Gated Potassium Channel Activities in Bovine Articular Chondrocytes.

Objectives. The objective of this paper is to determine if hyaluronan affects bupivacaine's anesthetic function. Methods. Whole cell patch clamp recordings were performed on bovine articular chondrocytes cultured in 60 mm dishes. The chondrocytes were treated with phosphate-buffered saline (control group), 7.5 mg/mL hyaluronan (Orthovisc), 0.25% bupivacaine, or a mixture of 7.5 mg/mL hyaluronan and 0.25% bupivacaine. Outward currents were elicited by step depolarization from -90 mV to 150 mV with 5 mV increments and holding for 200 ms. Results. The amplitude of outward currents elicited at 150 mV was 607.1 ± 135.4 pA (mean ± standard error) in the chondrocytes treated with phosphate buffered saline, 550.0 ± 194.9 pA in the chondrocytes treated with hyaluronan, 18.4 ± 8.3 pA in the chondrocytes treated with bupivacaine, and 12.8 ± 2.6 pA in the chondrocytes treated with a mixture of hyaluronan and bupivacaine. Conclusion. Hyaluronan does not affect bupivacaine's inhibitory action on the potassium channel activities in bovine articular chondrocytes. This finding suggests that intra-articular injection of a mixture of hyaluronan and bupivacaine may not affect the anesthetic effects of bupivacaine.

The roles of ionotropic glutamate receptors along the On and Off signaling pathways in the light-adapted mouse retina.

Although the locations of glutamate receptors along the On and Off pathways have been determined, how these receptors modulate the retinal outputs--the light-evoked and spontaneous activities of individual ganglion cells--is not fully understood in the mouse retina. Specifically, how these receptors mediate On and Off responses of retinal ganglion cells in mouse retina under light adaptation remains unknown. Since mouse retina has become a powerful model for vision research, the functions of glutamate receptors along the On and Off pathways in mouse need to be determined. In the current study, the light-evoked and spontaneous excitatory postsynaptic currents (light-evoked EPSCs and sEPSCs) from On, Off and On-Off retinal ganglion cells (RGCs) were recorded using whole-cell patch-clamp recordings to assess how NMDA and AMPA/KA receptors modulate the retinal outputs of RGCs in the light-adapted mouse retina. We found NMDA and AMPA/KA played different roles in light-evoked EPSCs along On and Off pathways in light-adapted mice retinas. Both NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP-5) and AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) acted on RGCs to reduce On responses of ganglion cells while they acted on Off-cone bipolar cells and/or ganglion cells to mediate Off responses of RGCs. Co-application of AP-5 and CNQX completely eliminated the Off responses in majority of RGCs, indicating that both NMDA and AMPA/KA receptors are critical for light signaling along the cone-driven Off pathways in the light-adapted mouse retina.

Retina protective effect of acidic fibroblast growth factor after canceling its mitogenic activity.

PURPOSE: The aim of this study was to investigate the effect of mutant of acidic fibroblast growth factor (MaFGF) on N-methyl-N-nitrosourea (MNU)-induced retinal degeneration in Sprague-Dawley rats. METHODS: Fifty (50)-day-old female Sprague-Dawley rats were given a single intraperitoneal injection of normal saline (NS) or 60 mg x kg(-1) body weight of MNU, and then NS or different doses of MaFGF were injected intravitreally twice at 0 and 12 h after NS or MNU treatment. After NS or MNU treatment for different times, the apoptotic index of the photoreceptor cell was detected by TUNEL labeling, whereas the mRNA expressions and the protein levels of antiapoptotic Bcl-2 and proapoptotic Bax were determined by reverse transcriptase polymerase chain reaction and Western blotting, respectively. Retinal damage was evaluated based on retinal thickness. RESULTS: MNU-induced retinal damage was partially protected by MaFGF in a dose-independent manner in rats. MaFGF at doses of 1.25 and 2.5 microg could partially suppress photoreceptor cell loss, whereas MaFGF at a dose of 5.0 mug had no protective effect on photoreceptor cell. The apoptotic index at 24 h post-MNU in the peripheral retina was 38.1 +/- 3.6%, whereas 1.25 and 2.5 mug MaFGF markedly reduced it to 27.5 +/- 2.0 and 21.1 +/- 1.9% (P = <0.001), respectively. As compared with the MNU-treated group, MaFGF significantly upregulated the expression of Bcl-2 mRNA and protein and downregulated the expression of Bax mRNA and protein (P = <0.001). CONCLUSION: MaFGF could counteract MNU-induced retinal damage and may be a therapeutic agent for the treatment of retinal degeneration.

Microarray expression analysis of the early N-methy-N-nitrosourea-induced retinal degeneration in rat.

The study was undertaken to investigate the gene expressions in N-methy-N-nitrosourea (MNU)-induced rat retinal degeneration (RD) by performing microarray analysis of retinal RNA at 12h. All rats were randomly divided into a normal group, a 12h model group and a 24h model group. Rats in the two model groups received a single intraperitoneal injection of 40 mg/kg body weight of MNU, while those in the normal group were injected with equivalent volume of physiological saline. After 12h and 24h of the injection, rats in each respective group were sacrificed, respectively. One eye of each animal was used for hematoxylin and erosin (H&E) staining, and fresh retinas of the other eye of each animal in the both normal group and 12h model group were used to extract total RNA, which was analyzed by microarray and real time RT-PCR. Retinal histological alteration was found in the 24h model group. There were 75 genes differently expressed (ratio > or =2.0), including 64 genes up-regulated and 11 genes down-regulated. Seven genes were assayed by real time RT-PCR and demonstrated the same alteration tendency as in microarray analysis. These genes that expressed differently mainly involved signal transduction, development, immune and defense, and apoptosis, etc. The major pathways were MAP-kinase signaling pathways, Toll-like receptor signaling pathway and apoptosis pathway involved. The results suggest that there are significant changes of gene expression in the early stage of MNU-induced RD. These microarray results provide clues to understand the molecular pathways underlying photoreceptor degeneration and indicate directions for future studies.

Protective effect of glycyrrhizin, glycyrrhetic acid and matrine on acute cholestasis induced by alpha-naphthyl isothiocyanate in rats.

Alpha-naphthyl isothiocyanate (ANIT) is a known hepatotoxicant that causes acute cholestatic hepatitis characterized by the infiltration of neutrophils around bile ducts and necrotic hepatocytes. The effects of glycyrrhizin (GL), 18beta-glycyrrhetinic acid (GA), matrine (MT), oxymatrine (OMT), salvianolic acid B (SAB), silymarin (SI) and dexamethasone (DEX) on ANIT-induced acute cholestasis in rats were investigated. Serological and histological data demonstrated that the administration of GL, GA or MT all protected against hepatocyte injury and cholestasis induced by ANIT. Furthermore, the bile flow and the accumulative bile excretion of ketoprofen glucuronide (KPG), that were significantly suppressed by ANIT, were preserved in rats administered GL, GA or MT. DEX protected against acute cholestasis but did not protect against hepatocyte necrosis and elevated serum alanine aminotransferase levels following ANIT administration. Rats administrated OMT, SAB or SI were not resistant to ANIT toxicity. In summary, the protective effect of DEX is directed toward cholangiocytes rather than hepatocytes whereas the natural products, GA, GL and MT, exhibit significantly better protective effects against ANIT-induced liver damage including the protection of hepatocytes as well as cholangiocytes.

Ketoprofen glucuronidation and bile excretion in carbon tetrachloride and alpha-naphthylisothiocyanate induced hepatic injury rats.

A pharmacokinetic study was carried out in rats to investigate the effects of experimental hepatic injury on the liver glucuronidation and bile excretion of ketoprofen (KP) and its glucuronides (KPGs). In vivo, KP (20mg/kg b.w.) was intravenously administered to carbon tetrachloride (CCl(4)) or alpha-naphthylisothiocyanate (ANIT) induced hepatic injury male rats. Concentrations of KP and its glucuronides (S-KPG and R-KPG) in plasma and bile were determined by RP-HPLC. It was observed that there was significant difference in the accumulative bile excretion of KPGs between the CCl(4) intoxicated rats and the normal rats (54+/-18.3% versus 90+/-6.9%), while it was extremely inhibited in ANIT intoxicated rats (2.0+/-3.1% versus 90+/-6.9%). As the result of reduction of KPGs excreted in bile, the area under the curve (AUC((0-infinity))) of KP and KPGs were higher in blood in CCl(4) and ANIT hepatic injury rats than those of the normal rats. Specifically, ANIT caused approximately 10-fold elevation of AUC((0-infinity)) of plasma S-KPG. In microsomal incubations experiment, the glucuronyltransferase activity was impaired in CCl(4) and ANIT intoxicated rats. It suggested that the glucuronyltransferase activity was impaired in CCl(4) and ANIT intoxicated rats, while the bile excretion function was suppressed extremely in ANIT intoxicated rats.

[Changes of NF-kappaB/I kappa B alpha in N-methyl-N-nitrosourea-induced retinal damage in rats].

OBJECTIVE: To observe the changes of nuclear factor-kappa B (NF-kappaB) in the course of N-methyl-N-nitrosourea (MNU)-induced apoptosis of rat retinal photoreceptor cells and investigate the mechanism of MNU-induced retinal damage. METHODS: A single intraperitoneal injection of 60 mg/kg MNU was given to 50-day-old female rats, which were sacrificed at different intervals after MNU treatment. The retinal damage was examined with optical microscopy and photoreceptor cell apoptosis detected by TUNEL assay. Western blotting was performed to analyze the changes in NF-kappaB. RESULTS: Pyknosis of the photoreceptor cell nuclei and disorientation of the outer segment of the photoreceptor layer was observed 24 h after MNU treatment, and the outer nuclear layer and photoreceptor layer were almost completely lost on day 7. Photoreceptor cell apoptosis peaked at 24 h, and in the apoptotic cascade, NF-kappaB p65 protein was only detected 12 and 24 h after MNU treatment, whereas the amount of I kappa B alpha, in contrast, markedly increased in the cytoplasm as well as in the nuclei. CONCLUSION: MNU-induced retinal damage might be mediated through the signaling pathway of NF-kappaB/I kappa B alpha.

[Protective effects of ligustrazine against photoreceptor cell injury induced by N-methyl-N-nitrosourea and its mechanism].

AIM: To study the protective effect of ligustrazine against photoreceptor cell injury induced by N-methyl-N-nitrosourea (MNU) in Sprague-Dawley (SD) rats. METHODS: Ligustrazine injections of different doses were injected intraperitoneally into 47-day female SD rats once a day and a single intraperitoneal injection of MNU 60 mg x kg(-1) was given to 50-day rats. At different intervals after MNU treatment,the animals were sacrificed. The apoptotic index of photoreceptor cells was calculated by TUNEL labeling at 24 h following MNU treatment; peripheral retinal damage was evaluated based on retinal thickness at the d 7 after MNU treatment, and the expression of c-jun and c-fos genes was detected by RT-PCR technique. RESULTS: Ligustrazine injection could remarkably increase total thickness of peripheral retina and decrease apoptotic index of photoreceptor cells induced by MNU in a dose-dependent manner. Compared with MNU-treated rats, the gene expression of c-jun and c-fos was time-dependently down-regulated in ligustrazine-treated group. CONCLUSION: Ligustrazine injection partially protects against MNU-induced retinal damage by down-modulating the expression of c-jun and c-fos genes to inhibit apoptosis of photoreceptor cells.

N-methyl-N-nitrosourea-induced apoptosis of photoreceptor cells in Sprague-Dawley rats via nuclear factor-kappaB.

BACKGROUND: Previous studies have showed that photooxidative stress can lead to down-modulation of nuclear factor-kappa B (NF-kappaB) activity causing apoptosis of cultured photoreceptor cells. This study aimed at investigating whether NF-kappaB was involved in photoreceptor cells apoptosis induced by N-methyl-N-nitrosourea (MNU) in rats. METHODS: A single intraperitoneal injection of 60 mg/kg MNU was given to 50-day-old female rats. At different intervals after MNU treatment, the animals were sacrificed. Retinal damage was examined by a light microscope. The apoptotic index of the photoreceptor cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). NF-kappaB was analysed by Western blot and Transcriptin Factor Assay Kits. RESULTS: The pyknosis of the photoreceptor nuclei and the disorientation of the outer segment of the photoreceptor layer was seen after MNU treatment for 24 hours. The outer nuclear layer and photoreceptor layer were almost completely lost at 7 days. Photoreceptor cells apoptosis reached the peaked value at 24 hours. In apoptotic cascade, the protein levels of NF-kappaB p65 were only detected after MNU treatment for 12 and 24 hours in the nucleus. Conversely, the amounts of IkappaBalpha were markedly increased in the cytoplasm as well as in the nucleus. The activity of NF-kappaB p65 in the nucleus was down-modulated in the end. CONCLUSIONS: MNU-induced photoreceptor cell destruction was attributed to the apoptotic process by down-regulating the activation of NF-kappaB p65.

[Protective effect of non-mitogenic haFGF on retinal injury induced by N-methyl-N-nitrosourea in Sprague-Dawley rats].

AIM: To study the effect of non-mitogenic human acidic fibroblast growth factor (nm-haFGF) on retinal injury induced by N-methyl-N-nitrosourea (MNU) in Sprague-Dawley rats and its mechanism. METHODS: Female rats of 50-days-old were injected with MNU (60 mg x kg(-1)) intraperitoneally, and three doses of nm-haFGF (1.25 microg, 2.5 microg and 5 microg in one eye of each rat) were injected, separately, into vitreous body of one eye of each rat twice a day at 0 and 12 h after MNU treatment. 24 h later, apoptotic index of photoreceptor cells was detected by TUNEL labeling and the expressions of Bcl-2 and Bax were analyzed by Western blotting. At the 7th day, retinal injury was evaluated based on retinal thickness. RESULTS: Compared with model group, apoptotic index of photoreceptor cells was significantly reduced in nm-haFGF groups at the dose of 1.25 microg and 2.5 microg in one eye of each rat at 24 h, and the total retinal thickness as well as the outer retinal thickness markedly increased 7 days after MNU, respectively. The expressions of Bcl-2 increased and that of Bax decreased adversely after being injected with different doses of nm-haFGF. CONCLUSION: nm-haFGF partially suppressed retinal injury induced by MNU in Sprague-Dawley rats. The mechanism could be related to up-regulation of Bcl-2 and down-regulation of Bax.

Tetramethylpyrazine protected photoreceptor cells of rats by modulating nuclear translocation of NF-kappaB.

AIM: To evaluate the effect of tetramethylpyrazine (TMP) injection on retinal damage induced by N-methyl-N-nitrosourea (MNU) in rats and on nuclear factor-kappa B (NF-kappaB) family members. METHODS: Female Sprague-Dawley (SD) rats were randomly divided into groups: (i), control group; (ii), model group; and (iii), TMP-injection groups, in which the rats were subdivided into 40 mg/kg, 80 mg/kg and 160 mg/kg groups. Drugs were injected ip into 47-day-old SD rats once a day. At 50 days of age, all rats in the model group and drug groups also received a single ip injection of 60 mg/kg MNU. Rats in group 1 received ip injection of physiological saline. All rats were killed at different times after MNU or physiological saline treatment. The apoptotic index of photoreceptor cells was calculated by TUNEL labeling; retinal damage was evaluated based on retinal thickness and the expression of NF-kappaB family members was detected by Western blot. RESULTS: TMP injections, in a dose-dependent manner, suppressed photoreceptor cell apoptosis and decreased its loss in the peripheral retina. As compared with the MNU-treated group, TMP injection at a dose of 160 mg/kg also time-dependently upregulated the NF-kappaB/p65 protein level in the nucleus and downregulated the IkappaBalpha protein level in the cytoplasm. However, no protective effect of TMP injection on MNU-induced central retinal damage was found. CONCLUSION: TMP injection partially protects against MNU-induced retinal damage by upregulating the nuclear translocation of p65 to inhibit photoreceptor cells apoptosis.

[The toxic effect of N-methyl-N- nitrosourea on retina in rats].

PURPOSE: To observe the toxic effect of N-methyl-N-nitrosourea (MNU) on photoreceptor cells within retina of SD rats. METHODS: At 50 days of age, 100 female rats received a single intraperitoneal injection of MNU at different doses of 50 mg/kg, 60 mg/kg, 70 mg/kg and 80 mg/kg body weight, respectively. Each group had 6 rats and 4 untreated rats were used as normal control. At 24, 48 and 72 hours and 7 days after the administration of MNU, the animals were sacrificed and both eyes were enucleated immediately and processed for histological examination. RESULTS: It was found that all doses of MNU could sequentially damage the central and peripheral retina. The first evidence of retinapathy 24 hours after the application of MNU was pyknosis and disruption of photoreceptor cells nuclei and the disorientation of the photoreceptor outer segments; loss of photoreceptor cell deteriorated significantly at 48 hours or 72 hours; the outer nuclear layer and photoreceptor layer were almost completely lost at 7 days. CONCLUSION: The results demonstrated that MNU could selectively damage the photoreceptor cells in the retina of the rats, which was dose-dependent and time-dependent.

[Antitumor effect of alcohol extract from Sesamum indicum flower on S180 and H22 experimental tumor].

OBJECTIVE: To investigate the antineoplasm effect of extract from Sesamum indicum L. flower. METHOD: Observing the effects of alcohol extract from Sesamum indicum flower on tumor growth in sarcoma 180 (S180) and Heps 22 (H22) tumorigenic mouse, and on weight of immune organs. RESULT: 6, 3, 1.5 g/kg extract showed inhibiting effect on tumor growth obviously, and had not distinct effect on weight of thymus and spleen in mice sarcoma 180 and Heps 22. CONCLUSION: The alcohol extract from Sesamum indicum flower showed obvious antitumor effect.

Caspases promoted DADAG-induced apoptosis in human leukemia HL-60 cells.

AIM: To investigate the roles of caspases in diacetyldianhydrogalactitol (DADAG)-induced apoptosis in human leukemia HL-60 cells. METHODS: Inhibition of proliferation was measured by MTT assay. DADAG-induced apoptosis in HL-60 cells was observed by electron microscopy, flow cytometry, and DNA fragmentation assay. Caspase-3 activity was determined by ApoAlert CPP32 colorimetric assay kit. The cleavage of substrates of caspases was detected by Western blot. RESULTS: DADAG exhibited potent antiproliferative activity and induced apoptosis in HL-60 cells. After treatment with DADAG 8 mg/L for 24 h, caspase-3 activity increased markedly and the cleavage of poly-(ADP-ribose) polymerase (PARP), lamin B, and DFF45 appeared. All of the apoptotic signals were suppressed by z-VAD fmk (a general inhibitor of caspase activities), whereas z-DEVD fmk, a selective inhibitor of caspase-3 activity, only induced partial reversion of the apoptotic effects. CONCLUSION: DADAG induced apoptosis in HL-60 cells by activating caspases. Caspases promoted apoptosis through the cleavage of substrates of PARP, lamin B, and DFF45.

[Apoptosis induced by diacetyldianhydrogalactitol and its mechanism in HL-60 leukemia cells].

AIM: To investigate the apoptosis induced by diacetyldianhydrogalactitol (DADAG) and its mechanism in human HL-60 leukemia cells. METHODS: Inhibition of proliferation was measured by MTT assay. DADAG-induced apoptosis in HL-60 cells was observed by electron microscopy, flow cytometry and DNA fragmentation assay. The levels of Bcl-2 family proteins were detected by Western blotting. Caspase-3 activity was determined by ApoAlert CPP32 colorimetric assay kit. RESULTS: DADAG exhibited potent antiproliferative activity and induced apoptosis in HL-60 cells. After treatment with DADAG 8 micrograms.mL-1 for various times, the Bcl-XL protein level decreased in a time-dependent manner, while the Bad protein level was upregulated. The caspase-3 activity increased markedly after treatment with DADAG for 24 h. The apoptotic signals were suppressed by z-VAD.fmk (a general inhibitor of caspases), whereas z-DEVD.fmk, a selective inhibitor of caspase-3, only induced partial reversion of the apoptotic effects. CONCLUSION: DADAG-induced apoptosis in HL-60 cells required caspase-3 activation and caspase-3 activation was related with Bcl-2 family members.