Search PubMed⌕ Search

Biomedical subjects

Y Gong

Publications and source records attributed to Y Gong.

At least 37 records · Page 2Linked to original sources
PubMed2003

Apolipoprotein E isoform-specific regulation of dendritic spine morphology in apolipoprotein E transgenic mice and Alzheimer's disease patients.

Dendritic spines are postsynaptic sites of excitatory input in the mammalian nervous system. Apolipoprotein (apo) E participates in the transport of plasma lipids and in the redistribution of lipids among cells. A role for apoE is implicated in regeneration of synaptic circuitry after neural injury. The apoE4 allele is a major risk factor for late-onset familial and sporadic Alzheimer's disease (AD) and is associated with a poor outcome after brain injury. ApoE isoforms are suggested to have differential effects on neuronal repair mechanisms. In vitro studies have demonstrated the neurotrophic properties of apoE3 on neurite outgrowth. We have investigated the influence of apoE genotype on neuronal cell dendritic spine density in mice and in human postmortem tissue. In order to compare the morphology of neurons developing under different apoE conditions, gene gun labeling studies of dendritic spines of dentate gyrus (DG) granule cells of the hippocampus were carried out in wild-type (WT), human apoE3, human apoE4 expressing transgenic mice and apoE knockout (KO) mice; the same dendritic spine parameters were also assessed in human postmortem DG from individuals with and without the apoE4 gene. Quantitative analysis of dendritic spine length, morphology, and number was carried out on these mice at 3 weeks, 1 and 2 years of age. Human apoE3 and WT mice had a higher density of dendritic spines than human E4 and apoE KO mice in the 1 and 2 year age groups (P<0.0001), while at 3 weeks there were no differences between the groups. These age dependent differences in the effects of apoE isoforms on neuronal integrity may relate to the increased risk of dementia in aged individuals with the apoE4 allele. Significantly in human brain, apoE4 dose correlated inversely with dendritic spine density of DG neurons cell in the hippocampus of both AD (P=0.0008) and aged normal controls (P=0.0015). Our findings provide one potential explanation for the increased cognitive decline seen in aged and AD patients expressing apoE4.

Aged↗
PubMed2003

Endogenous plasmin converts Glu-plasminogen to Lys-plasminogen on the monocytoid cell surface.

Recently, we showed that localization of Glu-plasminogen on cell surfaces enhances its conversion to Lys-plasminogen by exogenous plasmin. This leads to stimulation of plasminogen activation because Lys-plasminogen is the preferred substrate on cell surfaces. Here, we show that Glu-plasminogen was converted to Lys-plasminogen on monocytoid cells in the absence of exogenous plasmin. Culture of cells under serum-free conditions did not affect this conversion, suggesting that the enzymatic activity was cell-derived. Therefore, we tested whether endogenous monocytoid plasminogen could provide a source of plasmin to convert cell-associated Glu-plasminogen to Lys-plasminogen because plasmin is the only enzyme known to effect this reaction. We used a recombinant human plasminogen mutant, [D(646)E]Pg, which can be cleaved by plasminogen activators, but cannot catalyze the generation of Lys-plasminogen. Upon incubation with either THP-1 or U937 monocytoid cells, 35 and 38%, respectively, of the cell-bound ligand was converted to Lys-[D(646)E]Pg. Trasylol, alpha2-antiplasmin, and an anticatalytic antiplasminogen monoclonal antibody decreased Lys-[D(646)E]Pg formation to < 5% on monocytoid cells, consistent with a plasmin-dependent mechanism. Plasminogen was detected in these cells by Northern blotting and RT-PCR. Our results suggest that plasmin converts cell-bound Glu-plasminogen to Lys-plasminogen and that this enzyme is produced by activation of monocytoid plasminogen by endogenous monocytoid plasminogen activators to enhance plasminogen activation on the monocytoid cell surface.

Cell Line↗
PubMed2003

Glutathione S-transferase M1 and T1 polymorphisms and the risk of recurrent pregnancy loss.

The aetiology of recurrent pregnancy loss (RPL) remains unclear, but it may be related to a possible genetic predisposition together with involvement of environmental factors. We examined the relation between RPL and polymorphisms in two genes, glutathione S-transferases (GST) M1 and T1, which are involved in the metabolism of a wide range of environmental toxins and carcinogens. A case-control study of 115 cases with RPL and 160 controls was conducted. All cases and controls were women resident in Sapporo, Japan and the surrounding area. They were genotyped for polymorphisms of GSTM1 and GSTT1 using PCR-based methods. We found that 65.2% of the cases with RPL and 45.6% of the controls had the GSTM1 null genotype [odds ratio (OR) = 2.23, 95% confidence interval (CI) = 1.36-3.66]. On the other hand, 47.0% of the cases and 49.4% of the controls had the GSTT1 null genotype (OR = 0.95; 95% CI = 0.58-1.55). The results suggest that women with GSTM1 null polymorphism may therefore have an increased risk of RPL.

Abortion, Habitual↗
PubMed2003

Increasing cell membrane potential and GABAergic activity inhibits malignant hepatocyte growth.

Increasing hepatocyte membrane potentials by augmenting GABAergic activity inhibits nonmalignant hepatocyte proliferative activity. The objectives of this study were to document 1) potential differences (PDs) of four malignant hepatocyte cell lines, 2) GABAA receptor mRNA expression in the same cell lines, and 3) effects of restoring malignant hepatocyte PDs to levels approximating those of resting, nonmalignant hepatocytes. Hepatocyte PDs were documented in nonmalignant and malignant (Chang, HepG2, HuH-7, and PLC/PRF/5) hepatocytes with a fluorescent voltage-sensitive dye and GABAA receptor expression by RT-PCR and Western blot analyses. Compared with nonmalignant human hepatocytes, all four malignant cell lines were significantly depolarized (P < 0.0001, respectively). Only PLC/PRF/5 cells had detectable GABAA-beta3 receptor mRNA expression and all cell lines were negative for GABAA-beta3 receptor protein by Western blot analysis. Stable transfection of Chang cells with GABAA-beta3 receptor cDNA resulted in significant increases in PD and decreases in proliferative activity as manifest by decreased [3H]thymidine and bromodeoxyurieine incorporation rates, 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate activity, a lower mitotic index, prolongation of cell-doubling times, and attenuated growth patterns compared with cells transfected with vector alone. Colony formation in soft agar and the number of abnormal mitoses were also significantly decreased in GABAA-beta3 receptor transfected cells. The results of this study indicate 1) relative to healthy hepatocytes, malignant hepatocytes are significantly depolarized, 2) GABAA-beta3 receptor expression is absent in malignant hepatocyte cell lines, and 3) increasing the PD of malignant hepatocytes is associated with less proliferative activity and a loss of malignant features.

Carcinoma, Hepatocellular↗
PubMed2002

Phosphorylated tyrosine-containing proteins in primary lung cancer correlates with proliferation and prognosis.

To determine the usefulness of tyrosine phosphorylation in evaluating biological characteristics, we attempted to evaluate the relationship between the amount of phosphorylated tyrosine-containing proteins and clinicopathological factors, cell proliferation and outcome in non-small cell lung cancer. To evaluate phosphorylated tyrosine-containing proteins we used 96 surgically resected materials of non-small cell lung cancer and normal peripheral lung, while immunohistochemical evaluation was performed. Cell proliferating ability was evaluated using the labelling index of proliferating cell nuclear antigen-positive nuclear staining cells. There were statistically significant differences between the expression levels of phosphorylated tyrosine-containing proteins of normal and cancerous tissues (P<0.0001). Evaluations based on clinicopathological factors apart from histopathological differentiation, showed no statistically significant differences of phosphorylated tyrosine-containing proteins expression. However, phosphorylated tyrosine-containing proteins correlated with cell proliferation activity evaluated (P((Low, High))<0.0001; P((Low, Int)) <0.0001; P((Int, High))<0.0001). Furthermore, non-small cell lung cancer cases with high expression and intermediate expression of phosphorylated tyrosine-containing proteins had a significantly shorter disease-free postoperative survival than those with low expression of phosphorylated tyrosine-containing proteins using log-rank analysis (P((Low, Int)) <0.0028; P((Low, High))=0.0002). Furthermore, phosphorylated tyrosine-containing proteins expression level statistically contributed to disease-free survival in Cox's proportional hazard model. Therefore, phosphorylated tyrosine-containing proteins in non-small cell lung cancer tissues seem to reflect its biological malignancy, and this evaluation may be valuable for constructing the most appropriate therapeutic strategy.

Aged↗
PubMed2002

A cyclin-dependent kinase inhibitor (p21(WAF1/CIP1)) affects thymidine incorporation in human liver cancer cells.

p21(WAF1/CIP1) is a universal cyclin-dependent kinase inhibitor. To investigate the role of p21(WAF1/CIP1) in proliferation of human liver cancer cells, we examined the expression of p53, p21(WAF1/CIP1), cdk2 and cdk4 expression in two human liver cancer cell lines (HepG2 and PLC/PRF/5 cells). The effects of p21(WAF1/CIP1) on [(3)H]thymidine incorporation and cdks were also examined in these cells. HepG2 cells expressed all these proteins with lower level of cdk4. PLC/PRF/5 cells expressed the other proteins except p21(WAF1/CIP1). Transfection of p21(WAF1/CIP1) gene inhibited [(3)H]thymidine incorporation of both cells with different extent. Although the transfection of p21(WAF1/CIP1) did not affect cdk2 and cdk4 expression, it did reduce cdk2 kinase activity by 20%. These results suggest that: (a) p21(WAF1/CIP1) involved in DNA synthesis of human liver cancer cells; (b) p21(WAF1/CIP1) could be a target gene for the treatment of human hepatocellular carcinoma.

Adenosine Triphosphate↗
PubMed2002

Liver development in a rat model of fetal alcohol syndrome.

The inhibitory effects of alcohol on hepatic growth in adults raises the possibility that the liver may be involved in fetal alcohol syndrome (FAS) in infants. To test this hypothesis, pregnant Sprague-Dawley rats were fed liquid diets containing either ethanol as 36% of the total calories, or were allowed ad libitum feeding of a control liquid diet (controls) throughout pregnancy. Other dams were exposed to the ethanol diet only during the first or last half of pregnancy. Pups delivered of dams exposed to the various diets (N = 40-45/group) were killed at 1, 3, 7, and 14 days of age. In addition to brain weights, crown-rump lengths, and facial features, the following parameters of liver development were documented; liver weight, liver/body weight ratio, liver histology, hepatic ornithine decarboxylase activity (ODC), hepatic protein content, and rate of hepatic DNA synthesis (as determined by [3H]thymidine incorporation). The results revealed that pups exposed to ethanol throughout pregnancy but not ad libitum control diet pups had brain weights, crown-rump lengths, and facial features in keeping with FAS. With respect to liver development, the livers in FAS pups were consistently smaller than in the control group. However, total body weights were decreased to a greater extent, such that when corrected for body weights, the smaller livers in FAS pups only became significant on day 14 of life. Liver histology was similar in the two groups with no signs of active inflammation or fibrosis. Hepatic ODC activity was also similar, indicating no impairment in polyamine synthesis. Hepatic DNA synthesis rates were decreased in FAS pups at all time intervals. Pups delivered of dams exposed to ethanol during either the first or last half of pregnancy had results comparable to those of controls. To identify the mechanism(s) responsible for these findings, a second series of experiments was performed wherein the hepatic expression of the following factors associated with liver development were documented by northern-blot analyses; growth hormone receptor (GHr), insulin-like growth factor-I (IGF-I) and -II (IGF-II) and IGF binding proteins (IGFBPs) 1, 2, 3, and 4 mRNA on gestational days 16 and 20 and postpartum days 1 and 7. In this series, a third group of pups derived from dams in whom caloric consumption was matched to that of the ethanol-fed dams (isocaloric controls) were also studied. The results revealed no consistent differences in GHr, IGF, or IGFBP mRNA expression in the three groups. In conclusion, liver development and hepatic DNA synthesis were significantly impaired in this animal model of FAS. That impairment, however, was not associated with decreases in either polyamine synthesis or disturbances in the hepatic component of the GH/IGF/IGFBP axis.

Animals↗
PubMed2002

Proliferative and nutritional dependent regulation of glyceraldehyde-3-phosphate dehydrogenase expression in the rat liver.

Glyceraldehyde-3-phosphate dehydrogenase is a multifunctional protein possessing numerous cytoplasmic and nuclear functions associated with cellular proliferation. Despite the emerging role of glyceraldehyde-3-phosphate dehydrogenase in regulating the proliferative process, there is a paucity of data regarding its expression and intracellular distribution in non-malignant proliferating hepatocytes. Thus the aim of the present study was to document the intracellular distribution of glyceraldehyde-3-phosphate dehydrogenase protein in proliferating hepatocytes derived from regenerating rat livers, and glyceraldehyde-3-phosphate dehydrogenase gene expression in fasted and re-fed rats following partial hepatectomy (PHx). Glyceraldehyde-3-phosphate dehydrogenase mRNA and protein expression were documented by Northern and Western blot analyses, respectively, at various times following 70% PHx in adult Sprague-Dawley rats. At 24 h post-surgery, glyceraldehyde-3-phosphate dehydrogenase mRNA expression was significantly increased in both PHx and sham operated rats (P < 0.001), respectively. Despite the increase in glyceraldehyde-3-phosphate dehydrogenase mRNA expression in both groups, only PHx rats had a significant increase in the nuclear fraction of glyceraldehyde-3-phosphate dehydrogenase protein (threefold increase compared to sham and baseline levels, P < 0.01), cytoplasmic levels of glyceraldehyde-3-phosphate dehydrogenase protein remained unaltered in both groups. In terms of the effects of feeding and fasting on rats there were no significant differences in glyceraldehyde-3-phosphate dehydrogenase mRNA levels, whether fasted or refed, in rats that had undergone PHx, 8 h earlier. On the other hand, glyceraldehyde-3-phosphate dehydrogenase mRNA levels were significantly increased in refed compared to fasted sham operated rats 8 h following surgery. Serum insulin concentrations were higher in the refed PHx and sham groups compared to their fasted counterparts. The results of this study indicate that although glyceraldehyde-3-phosphate dehydrogenase mRNA are altered to the same extent in PHx and sham-operated rats following surgery, increases in the nuclear fraction of glyceraldehyde-3-phosphate dehydrogenase protein only occur in PHx rats. The results also indicate that glyceraldehyde-3-phosphate dehydrogenase expression is affected by the nutritional status of animals undergoing abdominal sham surgery.

Animals↗
PubMed2002

Localization of major gangliosides in the PNS: implications for immune neuropathies.

Antibodies targeting major gangliosides that are broadly distributed in the nervous system are sometimes associated with clinical symptoms that imply selective nerve damage. For example, anti-GD1a antibodies are associated with acute motor axonal neuropathy (AMAN), a form of Guillain-Barré syndrome that selectively affects motor nerves, despite reports that GD1a is present in human axons and myelin and is not expressed differentially in motor versus sensory roots. We used a series of high-affinity monoclonal antibodies (mAbs) against the major nervous system gangliosides GM1, GD1a, GD1b and GT1b to test whether any of them bind motor or sensory fibres differentially in rodent and human peripheral nerves. The following observations were made. (i) Some of the anti-GD1a antibodies preferentially stained motor fibres, supporting the association of human anti-GD1a antibodies with predominant motor neuropathies such as AMAN. (ii) A GD1b antibody preferentially stained the large dorsal root ganglion (DRG) neurones, in keeping with the proposed role of human anti-GD1b antibodies in sensory ataxic neuropathies. (iii) Two mAbs with broad structural cross-reactivity bound to both gangliosides and peripheral nerve proteins. (iv) Myelin was poorly stained; all clones stained axons nearly exclusively. Our findings suggest that anti-ganglioside antibody fine specificity as well as differences in ganglioside accessibility in axons and myelin influence the selectivity of injury to different fibre systems and cell types in human autoimmune neuropathies.

Animals↗
PubMed2001

Influence of angiogenetic factors and matrix metalloproteinases upon tumour progression in non-small-cell lung cancer.

We attempted to investigate immunohistochemical expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PD-ECGF), c-erbB-2, matrix metalloproteinase-2 (MMP-2), and MMP-9 using surgical specimens of 119 non-small-cell lung carcinoma (NSCLC) cases and to evaluate the relationship between the expression levels of each molecule and clinicopathological factors or prognosis. VEGF expression levels were significantly associated with the local invasion (P = 0.0001), lymph node involvement (pN-factor) (P = 0.0019), pathological stage (p-stage) (P = 0.0027) and lymphatic permeation (P = 0.0389). PD-ECGF expression levels were associated with pN-factor (P = 0.0347). MMP-2 expression levels were associated with pN-factor (P = 0.004) and lymphatic permeation (P = 0.0056). Also, MMP-9 expression levels showed a significant correlation to local invasion (P = 0.0012), pN-factor (P = 0.0093) and p-stage (P = 0.0142). Multivariate analysis showed VEGF to be the most related to local invasion (P = 0.0084), and MMP-2 was the only factor with significant independent impact on lymphatic permeation (P = 0.0228). Furthermore, log-rank analysis showed significant association with poor survival by VEGF, bFGF, MMP-2 and MMP-9. Especially, combined overexpression of VEGF and MMP-2 revealed poor prognosis, our study might provide a basis for the better evaluation of biological characteristics and a new therapeutic strategy based on chemotherapy.

Adult↗
PubMed2001

The murine Wilms tumor suppressor gene (wt1) locus.

The Wilms tumor suppressor gene WT1 plays a crucial role in the etiology of various human diseases as well as in the development of specific organs including the kidneys, gonads and the spleen. At present the human as well as the Fugu wt1 locus have been characterized. We have used a PAC clone to analyze the murine wt1 locus and report here the structure of the wt1 gene as well as a characterization of the nine wt1 introns regarding their size and sequence at the exon/intron and intron/exon boundaries. In addition we provide a restriction map of the murine wt1 locus which should prove useful for the cloning of various constructs designed for the generation of mouse models. Prompted by the existence of a WT1 antisense transcript in humans we also examined strand-specific transcription at the murine wt1 locus. Our analysis suggests that there is no detectable antisense transcription of sequences within or immediately downstream of wt1 exon 1. We find, however, evidence for a divergent transcript which encompasses sequences at and around minor transcriptional initiation sites of wt1 and which is transcribed in the opposite direction. Despite the very high degree of similarity between the human and the murine wt1 sequence and expression as well as the presence of divergent transcripts in both cases, the existence of antisense transcription does not seem to be conserved between the two species.

Amino Acid Sequence↗
PubMed2001

LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development.

In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). We find that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. We demonstrate LRP5 expression by osteoblasts in situ and show that LRP5 can transduce Wnt signaling in vitro via the canonical pathway. We further show that a mutant-secreted form of LRP5 can reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Pharmacological characterization of a novel factor Xa inhibitor, FXV673.

FXV673 is a novel, potent, and selective factor Xa (FXa) inhibitor. FXV673 inhibited human, dog, and rabbit FXa with a K(i) of 0.52, 1.41, and 0.27 nM, respectively. FXV673 also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C (aPC), plasmin, and tissue-plasminogen activator (t-PA). FXV673 prolonged plasma activated partial thromboplastin time (APTT) and prothrombin time (PT) in a dose-dependent fashion. In the APTT assays, the concentrations (microM) required for doubling coagulation time were 0.41 (human), 0.65 (monkey), 1.12 (dog), 0.25 (rabbit), and 0.80 (rat). The concentrations (microM) required in the PT assays were 1.1 (human), 1.32 (monkey), 2.31 (dog), 0.92 (rabbit), and 1.69 (rat). A coupled-enzyme assay was performed to measure thrombin activity following prothrombinase conversion of prothrombin to thrombin. FXV673 showed IC(50)s of 1.38 and 2.55 nM, respectively, when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or fresh platelets were used as the phospholipid source for prothrombinase complex formation. It was demonstrated that FXV673 could inhibit further thrombin generation in the prothrombinase complex using PS/PC liposomes. FXV673 dose-dependently prolonged the time to vessel occlusion and inhibited thrombus formation in well-characterized canine models of thrombosis. Interspecies extrapolation (approximately 2.5-fold higher sensitivity for FXa inhibition in human than in dog) suggested that 100 ng/ml of FXV673 would be an effective plasma concentration for clinical studies. Currently FXV673 is undergoing clinical studies to be developed as an antithrombotic agent.

Animals↗
PubMed2001

Evidence that the RNAseH activity of the duck hepatitis B virus is unable to act on exogenous substrates.

BACKGROUND: The hepadnaviral reverse transcriptase can synthesize DNA on its native RNA template within viral cores but it is usually unable to synthesize DNA employing exogenous nucleic acids as a template. The mechanism of this template commitment is unknown. Here we provide evidence that the RNAseH activity of duck hepatitis B virus reverse transcriptase may also be unable to act on exogenous substrates. RESULTS: RNAseH assays were performed under a wide variety of conditions employing substrate RNAs of Duck Hepatitis B Virus sequence annealed to complementary DNA oligonucleotides and permeabilized intracellular viral core particles. Temperature, pH, cation type, salt concentration, substrate concentration, and the sequences of the cleavage sites were varied, and the effects of ATP and dNTPs on RNAseH activity were examined. duck hepatitis B virus RNAseH activity was not detected under any of these conditions, although E. coli or Avian Myeloblastosis Virus RNAseH activity could be detected under all conditions. Access of the RNA substrate to the enzyme within the viral cores was confirmed. CONCLUSIONS: These results imply that the RNAseH activity of the DHBV reverse transcriptase may not be able to degrade exogenous RNA:DNA heteroduplexes, although it can degrade heteroduplexes of the same sequence generated during reverse transcription of the endogenous RNA template. Therefore, the RNAseH activity appears to be "substrate committed" in a manner similar to the template commitment observed for the DNA polymerase activity.

Hepatitis B Virus, Duck↗
PubMed2001

Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells.

With the goal of identifying genes that have an expression pattern that can facilitate the diagnosis of primary breast cancers (BCs) as well as the discovery of novel drug leads for BC treatment, we used cDNA hybridization arrays to analyze the gene expression profiles (GEPs) of nine weakly invasive and four highly invasive BC cell lines. Differences in gene expression between weakly and highly invasive BC cells were identified that enabled the definition of consensus GEPs for each invasive phenotype. To determine whether the consensus GEPs, comprising 24 genes, could be used to predict the aggressiveness of previously uncharacterized cells, gene expression levels and comparative invasive and migratory characteristics of nine additional human mammary epithelial cell strains/lines were determined. The results demonstrated that the GEP of a cell line is predictive of its invasive and migratory behavior, as manifest by the morphology of its colonies when cultured on a matrix of basement membrane constituents (i.e., Matrigel). We found that the expression of keratin 19 was consistently elevated in the less aggressive BC cell lines and that vimentin and fos-related antigen-1 (FRA-1) were consistently overexpressed in the more highly aggressive BC cells. Moreover, even without these three genes, the GEP of a cell line still accurately predicted the aggressiveness of the BC cell, indicating that the expression pattern of multiple genes may be used as BC prognosticators because single markers often fail to be predictive in clinical specimens.

Animals↗
PubMed2001

Reactive oxygen species involved in trichosanthin-induced apoptosis of human choriocarcinoma cells.

The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumour and anti-HIV activities. We have found for the first time that TCS stimulated the production of reactive oxygen species (ROS) in JAR cells (a human choriocarcinoma cell line) in a time- and concentration-dependent manner by using the fluorescent probe 2',7'-dichlorofluorescein diacetate with confocal laser scanning microscopy. ESR spectral studies and the inhibition of ROS formation by the superoxide radical anion (O(2)(-.)) scavenger superoxide dismutase, the H(2)O(2) scavenger catalase and the hydroxyl radical (OH(.)) scavenger mannitol suggested the involvement of O(2)(-.), H(2)O(2) and OH(.). TCS-induced ROS formation was shown to be dependent on the presence of both extracellular and intracellular Ca(2+); moreover, ROS production paralleled the intracellular Ca(2+) elevation induced by TCS, suggesting that ROS production might be a consequence of Ca(2+) signalling. TCS-induced activation of caspase-3 was initiated within 2 h; however, TCS-induced production of ROS was initiated within 5 min, suggesting that the production of ROS preceded the activation of caspase-3. Simultaneous observation of the nuclear morphological changes via two-photon laser scanning microscopy and ROS production via confocal laser scanning microscopy revealed that ROS is involved in the apoptosis of JAR cells. The involvement of ROS was also confirmed by the inhibition of TCS-induced cell death by the antioxidant Trolox and the ROS scavengers catalase and mannitol. Diethylenetriaminepenta-acetic acid, an inhibitor of metal-facilitated OH(.) formation, markedly inhibited TCS-induced cell death, suggesting that TCS induced OH(.) formation via the Fenton reaction. The finding that ROS is involved in the TCS-induced apoptosis of JAR cells might provide new insight into the anti-tumour and anti-HIV mechanism of TCS.

Antineoplastic Agents, Phytogenic↗