Bryostatin 1 induces ubiquitination and proteasome degradation of Bcl-2 in the human acute lymphoblastic leukemia cell line, Reh

The ubiquitin-mediated proteolytic system has been implicated in the turnover of a number of intracellular proteins. In the present study, we investigated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquitin-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and immunoblotting analyses revealed that Bcl-2 is ubiquitinated following exposure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryostatin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the control after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 protein again rapidly decreases to 6% of its pre-bryostatin 1 level at which time a plateau is reached and maintained for another 72 h. Furthermore, ubiquitin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 treated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of Bcl-2. However, ubiquitin-Bcl-2 conjugates increase in a time-dependent manner following bryostatin 1 treatment. Lactacystin, which inhibits the proteinase activities of the proteasome, inhibited the bryostatin 1-induced decrease of Bcl-2 protein. The effect of bryostatin 1 on the proteolytic efficiency of the 26S proteasome in Reh cell extracts was also investigated and shown to increase following 1 h of bryostatin 1 treatment. Proteolytic activity reached its highest point by 3 h, and subsequently returned to control levels by 12 h, post-bryostatin 1 treatment. In addition, bryostatin 1 treatment of the Reh cell line decreased expression of bcl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. We speculate that this decrease in mRNA together with increased 26S proteolytic activity accounts for the initial rapid decrease recorded in Bcl-2 protein. These findings indicate that bryostatin 1 treatment of Reh ALL cells decreases Bcl-2 expression through two processes: a) enhanced Bcl-2 protein degradation through the activation of the ubiquitin-proteasome pathway and b) decreased bcl-2 mRNA expression.     

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.     

Factors affecting bryostatin 1-enhanced 2-CdA cytotoxicity in resistant B-cell chronic lymphocytic leukemia

Pre-treatment with bryostatin 1 (bryo) has been shown to potentiate the efficacy of (2-chloro-2-deoxyadenosine, cladribine, 2-CdA) in B-cell chronic lymphocytic leukemia (B-CLL) by increasing the ratio of deoxycytidine kinase (dCK) to 5'-nucleotidase (5'-NT) activity. The bryo-induced increase in dCK/5'-NT activity alone has not been a conclusive indication of final clinical outcome. Therefore, we used an ex vivo assay to investigate factors which may affect the bryo-induced enhancement of 2-CdA efficacy in B-CLL patient-derived samples. Bryo-induced increase in dCK/5'-NT was inversely associated with Rai stage CLL (r=-0.86). Increased dCK/5'-NT activity was not correlated with increased efficacy (cell death) or percentage of cellular [8-3H]-2-CdA converted to [8-3H]-2-CdATP ex vivo. Bryo pre-treatment increased the cellular uptake of [8-3H]-2-CdA and incorporation of [8-3H]-2-CdA metabolites into the DNA fraction. Cell death from 2-CdA was inversely correlated with bryo-induced activity of the DNA repair enzyme, DNA-PKcs, (r=-0.77). Thus, the ability of B-CLL to repair damaged DNA may be a more important predictor of the response to bryo/2-CdA and eventual clinical outcome than dCK/5'-NT activity. Additional CLL patients under bryo-2-CdA therapy are needed to verify these important observations.     

Downregulation of glutamate transporters is associated with elevation in extracellular glutamate concentration following rat microsphere embolism

Sodium-dependent glutamate transporters expressed in astroglial cells and neurons are essential for clearance of extracellular glutamate. In the present study, we found elevation of extracellular glutamate concentration associated with concomitant downregulation of glutamate transporters following rat microsphere embolism (ME). A marked increase in extracellular glutamate in the rat striatum was observed by microdialysis immediately after ME induction, and glutamate remained elevated at least 12 h after ischemia. Concomitantly, impairment of high KCl (146 mM)-induced glutamate release was observed in the striatum 12 h after ME. Consistent with the persistent increase in extracellular glutamate, expression of the glutamate transporters EAAC1 and GLT-1 significantly decreased 6 h after insult without a change in GLAST levels. GLT-1 expression was restored to basal levels within 48 h, whereas EAAC1 expression remained decreased up to at least 72 h after ME. Restoration of GLT-1 was associated with increased expression of the astroglial marker GFAP, whereas markedly reduced EACC1 levels were correlated with reduced levels of the neuronal marker MAP2, likely due to loss of vulnerable neurons. Taken together, downregulation of glutamate transporters after ME is associated with dysregulation of basal glutamate concentrations and KCl-induced glutamate release in the brain.     

Cisplatin-induced apoptosis in cultures of human Schwann cells

To investigate the sensitivity of human Schwann cells to cisplatin (cis-DDP), different approaches to estimate DNA damage were used: the comet assay, morphological evaluation of the granular condensation of nuclear chromatin and the terminal transferase-mediated dUTP nick-end-labelling (TUNEL) method. The number of micronuclei (MNi), as a sign of cisplatin-induced genotoxicity, was counted. DNA damage assessed by the comet assay was already evident after 1.5 microM cisplatin treatment at all exposure times (24, 48, and 72 h). Initial morphological changes characterised by the granular condensation of nuclear chromatin were detectable after 24 h exposure to 25 microM cis-DDP, while an increased number of apoptotic cells, determined by the TUNEL method, was noted after 48 h exposure to the same concentration. The first significant increase in the number of MNi was observed in cells treated with 75 microM cis-DDP for 24 h. We demonstrate that the comet assay is a highly sensitive method for measuring cisplatin induced DNA damage. Morphological observation revealed advanced as well as less prominent alterations in the nuclear chromatin. In contrast, the TUNEL method detected only those cells with advanced DNA fragmentation.     

Post-exposure targeting of specific epitopes on ricin toxin abrogates toxin-induced hypoglycemia, hepatic injury, and lethality in a mouse model

Effects in the liver of fatal intoxication with the binary toxin ricin are unclear. We report a robust neutrophil influx into the liver of C57BL/6 mice after lethal parenteral ricin challenge, occurring in peri-portal and centro-lobular hepatic areas within 2 h, followed by the abrupt disappearance of hepatic macrophages/Kupffer cells. Chemokine profiles determined by microarray, ribonuclease protection assays, northern blotting, and enzyme-linked immunosorbent assays showed rapid (2 h) upregulation and persistence of those for neutrophils (CXCL1/KC, CXCL2/MIP-2) and monocytes (CCL2/MCP-1). Red blood cell pooling (8-12 h), loss of hepatocyte glycogen (8-48 h) associated with progressive hypoglycemia, fibrin deposition (24-48 h), and death (72-96 h) followed. Monoclonal antibody to ricin A chain, administered intravenously, blunted hypoglycemia, and abrogated death. This outcome was observed when anti-ricin antibody was given before toxin exposure as well as when administered approximately 10 h after toxin exposure. Targeting antibody to specific amino-acid sequences on the ricin A chain (HAEL and QXXWXXA) was critical to the therapeutic effect. Re-emergence of liver macrophages/Kupffer cells and replenishment of glycogen in previously depleted hepatocytes preceded full recovery of the host. These data identify critical events for liver injury and healing in ricin intoxication, as well as a new means and specific targets for post-exposure therapeutic intervention.     

Intracerebral injection of autologous whole blood in rats: time course of inflammation and cell death

Intracerebral hemorrhage is associated with stroke and head trauma. The purpose of this study was to study brain inflammation and cell death in adult rats 1 h to 4 weeks after injection of blood into the striatum. Terminal dUTP nick-end-labeling positive dying cells were evident 4 h to 4 weeks post-hemorrhage. Neutrophil infiltration was brief and peaked at 48 h. CD8a immunoreactive lymphocytes, possibly natural killer cells, became apparent at 48 h and persisted for 1 week. Microglial reaction was evident at 4 h and persisted for 4 weeks. We conclude that extravascular blood causes a mixed inflammatory cell reaction in brains that is maximal from 48-72 h following hemorrhage. This is associated with death of brain cells over a prolonged period of at least 4 weeks.     

Increased accumulation of cytosine arabinoside in human leukemic cells and enhancement of its cell-killing activity by uridine

The effects of uridine(UR) on the cell-killing activity of cytosine arabinoside(ara-C) against human leukemic cells, MOLT-4, and on ara-C accumulation in cells were studied. The 50% lethal dose(LD50) of ara-C as determined by clonogenic assay was decreased to 5.0 x 10(-8) mol from 9.0 x 10(-7) mol after 3 days exposure to 10(-3) mol of UR. The accumulation of 3H-ara-C at 24 and 48 h was significantly increased in culture medium containing 10(-8) mol of 3H-ara-C and 10(-3) mol of UR (5,129 +/- 123.5 vs 2,554 +/- 115.5 cpm/10(5) cells at 24 h, p less than 0.01, and 5,772 +/- 123.2 vs 1,372 +/- 51.8 cpm/10(5) cells at 48 h, p less than 0.01). It is noteworthy that cell-killing activity of ara-C against human leukemic cells was enhanced by the combination with a nucleoside(UR), but not with antileukemic agents.     

Ozone-induced acute pulmonary injury in inbred mouse strains

To determine if host factors influence the time course and extent of lung injury after acute inhalation of ozone (O3), we evaluated the physiologic and biologic response of nine genetically diverse inbred strains of mice (C57BL/6J, 129/SvIm, BTBR, BALB/cJ, DBA/2J, A/J, FVB/NJ, CAST/Ei, and C3H/HeJ) exposed to O3 (2.0 ppm x 3 h). Whole lung lavage determined that 129/Svlm, BTBR, DBA/2J, and FVB/NJ had a peak increase in polymorphonuclear cells (PMNs) at 6 h, whereas C57BL/6J and CAST/Ei had a peak increase at 24 h after exposure; airway PMNs were minimally elevated in A/J and C3H/HeJ; BALB/cJ had a predominant lymphocytic influx. Interleukin-6 concentration in the lavage fluid was associated with the influx of PMNs, whereas the total protein in the lavage fluid did not always correlate with lavage cellularity. Respiratory responses were monitored using whole body plethysmography and enhanced pause index. C57BL/6J, BALB/cJ, 129/SvIm, and BTBR were highly sensitive to O3 and exhibited significant increases in enhanced pause to methacholine aerosol stimulation at 6 and 24 h after exposure to O3. In contrast, DBA/2J, A/J, FVB/NJ, CAST/Ei, and C3H/HeJ strains had demonstrated increases in sensitivity to MCh at 6 h after exposure, but responses had returned to near baseline by 24 h after exposure to O3. Epithelial cell proliferation as assessed by proliferating cell nuclear antigen staining was evident at 24 h after exposure to O3. C57BL/6J and A/J showed 4% proliferating cell nuclear antigen-positive cells; 129/SvIm, DBA/2J, and FVB/NJ had 1-3%; and BTBR, BALB/cJ, CAST/Ei, and C3H/HeJ had < 1%. Phenotypic measurements in six inbred strains were used for an in silico genome analysis based on the Roche mouse database. Consistent loci on chromosomes 1, 7, and 15 were among those identified to have a significant association with the phenotypes studied. In aggregate, our approach has identified O3-resistant (C3H/HeJ and A/J) and -vulnerable (C57BL/6J and 129/SvIm) strains of mice, and determined novel genomic loci, suggesting a clear genetic basis for the lung response to inhaled O3.     

Cisplatin-DNA damage in p21WAF1/Cip1 deficient mouse keratinocytes exposed to cisplatin

In response to DNA damage, cell cycle arrest, apoptosis, and DNA repair are mediated by a TP53 pathway that induces p21(WAF1/Cip1). The chemotherapeutic drug cis-diamminedichloroplatinum-II (cisplatin) damages cellular DNA by forming cis-diammineplatinum-N(7)-d[GpG] and cis-diammine-platinum-N(7)-d[ApG] adducts. To investigate the role of p21, skin keratinocytes from p21(WAF1/Cip1) wild-type (+/+), heterozygous (+/-), and null (-/-) mice, cultured in calcium levels designed to maintain a proliferating state, were exposed to 5 microM cisplatin continuously for 0, 8, 24, 48 and 72 h. At all time points the (+/-) cells had the fewest Pt-DNA adducts, and at 24 h mean Pt-DNA adduct levels were 541, 153 and 779 fmol adduct/mug DNA for p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively [P < 0.05 for (+/+) versus (+/-) and (-/-) versus (+/-)]. In order to understand underlying events, we examined p21(WAF1/Cip1) messenger RNA (mRNA), cell cycle arrest, and apoptosis in these cells. At 48 h of cisplatin exposure p21(WAF1/Cip1) mRNA expression was 2-fold higher in the (+/+) cells, compared to the (+/-) cells. At 24 h, the % of cells in S-phase in cisplatin-exposed cultures, compared to unexposed cultures, was decreased by 51, 40 and 11% in p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively (P = 0.04, ANOVA). At 24, 48 and 72 h the % of cisplatin-exposed (+/+) cells in apoptosis was 9.4-10.5%, while the cisplatin-exposed (+/-) and (-/-) cells had 1.2-3.7% of cells in apoptosis. The data support the interpretation that DNA replication arrest and apoptosis do not completely explain the low levels of Pt-DNA adducts in the (+/-) cells, and suggest that p21(WAF1/Cip1) controls activity resulting in either low Pt-DNA adduct formation or enhanced Pt-DNA adduct removal.     

Protective role of IL-2 during activation of T cells with bryostatin 1

Pharmacologic agents such as bryostatin 1 (bryostatin) can regulate cell activation, growth, and differentiation by modulating the activities of protein kinase C isoenzymes. Inhibition of growth of tumor cells and activation of T lymphocytes in vitro are the most recognized consequences of bryostatin treatment. The effect of bryostatin on T cells ranges from induction of apoptotic cell death to T cell activation, expansion, and acquisition of antigen-specific effector functions. Here, we describe the conditions under which these wide ranging effects occur. Mouse mammary tumor 4TO7-IL-2-primed lymph node cells exposed ex vivo to bryostatin upregulated CD25 expression but lost the ability to secrete IL-2. Most of these cells died by apoptosis unless IL-2 was provided for the duration of bryostatin treatment. Analysis of T cell repertoire by screening of T cells for the expression of different Vbeta T cell receptor (TCR) families revealed that bryostatin-induced T cell death was unbiased and Vbeta-nonspecific. Within particular Vbeta clones, only CD25(+) T cells survived exposure to bryostatin and IL-2. Treatment of 4TO7 tumor-bearing mice with a single injection of low dose bryostatin followed by multiple low doses of IL-2, but not with bryostatin alone, delayed tumor growth. These results indicate that activation of T cells with bryostatin should be carried out under protection of exogenous IL-2 to ensure survival and expansion of T cells that may exhibit anti-tumor activity.     

Pulmonary mechanics and lung histology in acute lung injury induced by Bothrops jararaca venom

Pulmonary mechanics [static (Est) and dynamic (Edyn) elastances, resistive (DeltaP1) and viscoelastic pressures (DeltaP2)], histology, and bronchoalveolar lavage fluid (BALF) from BALB/c mice were analysed 1, 24, 48 and 72 h after intravenous injection of saline or Bothrops jararaca crude venom [0.3 (V0.3) or 1 (V1) microg.g(-1)]. Est, Edyn, and DeltaP2 increased at 1 h in both V groups, being significantly higher in V1 than in V0.3, decreasing progressively, reaching control values at 48 h in V0.3, but remaining altered in V1 at 72 h. DeltaP1 augmented in V1 at 1 h, returning to normal at 72 h. Histological changes in V0.3 group included interstitial oedema, alveolar collapse, and increased cellularity, which returned to normal at 48 h. These changes were more intense in V1 group, with alveolar oedema and haemorrhage. BALF showed time-dependent neutrophil influx in V0.3. In conclusion, venom led to time- and dose-dependent pulmonary mechanical changes, together with moderate inflammation in V0.3 and acute lung injury in V1.     

Role of the D2 dopamine receptor in molecular adaptation to chronic hypoxia in PC12 cells

We have previously shown that pheochromocytoma (PC12) cells rapidly depolarize and undergo Ca2+ influx through voltage-dependent Ca2+ channels in response to moderate hypoxia and that intracellular free Ca2+ is modulated by activation of dopamine D2 receptors in this cell type. The present study shows that D2 (quinpirole-mediated) inhibition of a voltage-dependent Ca2+ current (ICa) in PC12 cells is dramatically attenuated after chronic exposure to moderate hypoxia (24 h at 10% O2). Pretreatment of cells with pertussis toxin abolished D2-mediated inhibition of ICa. The D2-induced inhibition of ICa did not depend on protein kinase A (PKA), as it persisted both in the presence of a specific PKA inhibitor (PKI) and in PKA-deficient PC12 cells. Prolonged exposure to hypoxia (24 h) significantly reduced the level of Gi/o alpha immunoreactivity, but did not alter G beta levels. Furthermore, dialysis of recombinant G(o) alpha protein through the patch pipette restored the inhibitory effect of quinpirole in cells chronically exposed to hypoxia. We conclude that the attenuation of the D2-mediated inhibition of ICa by chronic hypoxia is caused by impaired receptor-G protein coupling, due to reduced levels of G(o) alpha protein. This attenuated feedback modulation of ICa by dopamine may allow for a more sustained Ca2+ influx and enhanced cellular excitation during prolonged hypoxia.     

p38 MAPK-iNOS-NO通路介导化学性缺氧对PC12细胞的损伤作用

目的:探讨诱导型一氧化氮合酶/一氧化氮(iNOS/NO)是否介导了氯化钴(CoCl2)引起的PC12细胞损伤及p38 MAPK对其调节作用。方法:应用化学性低氧模拟剂CoCl2处理PC12细胞建立化学性缺氧损伤模型。应用细胞计数试剂盒-8(CCK-8)比色法检测细胞存活率;Hochest33258核染色法观察细胞凋亡的形态学改变;Western blotting法检测iNOS蛋白的表达水平;Griess试剂盒检测细胞培养基中亚硝酸盐(NO的代谢物)的浓度。结果:应用600μmol/L CoCl2处理PC12细胞24 h可使iNOS表达明显增多;应用600μmol/L CoCl2处理PC12细胞24 h和48 h可使细胞培养基里NO增多;在CoCl2损伤PC12细胞前60 min应用iNOS抑制剂L-canavanine(10μmol/L)预处理能保护PC12细胞对抗600μmol/L CoCl2引起的损伤,使细胞存活率升高,凋亡细胞数目减少;SB203580(p38 MAPK选择性抑制剂)预处理60 min可下调CoCl2引起的iNOS高表达。结论:p38 MAPK-iNOS-NO通路介导CoCl2引起PC12细胞的损伤作用。     

Lipopolysaccharide-induced down-regulation of Ca2+ release-activated Ca2+ currents (I CRAC) but not Ca2+-activated TRPM4-like currents (I CAN) in cultured mouse microglial cells.

Microglia are the main immunocompetent cells of the mammalian central nervous system (CNS). Activation of cultured microglial cells and subsequent release of nitric oxide and cytokines critically depends on intracellular calcium levels. Since microglia undergo dramatic morphological, biochemical and electrophysiological changes in response to pathological events in the CNS, we investigated temporal changes in expression levels of ion channels involved in cellular calcium homeostasis in mouse cortical microglial cells in culture. Specifically, we assessed the inward and delayed outward rectifier potassium currents (I IRK and I DRK), calcium (Ca2+) release-activated Ca2+ currents (I CRAC) and Ca2+-activated TRPM4-like currents (I CAN) in non-activated microglia and cells that were activated by exposure to lipopolysaccharide (LPS) between 3 and 48 h. Unstimulated microglial cells, subcultured from an astrocyte coculture, typically exhibited a ramified, rod-shaped morphology. During the first 3 days of culture cell size and shape were maintained, but the percentage of cells showing prominent I IRK went up and those expressing I DRK went down. Cells retaining I DRK exhibited smaller amplitudes, whereas those of I IRK and I CRAC were not affected. However, after 24 h of exposure to 1 microg ml(-1) LPS, most cells showed an amoeboid ('fried egg'-shaped) morphology with a 62% increase in cell capacitance. At that point in time, only 14% of the cells revealed I IRK and 3% had I DRK exclusively, whereas the majority of cells expressed both currents. The amplitudes of I CRAC and I IRK progressively decreased after stimulation, whereas I DRK transiently reached a maximum after 6 h of LPS exposure and then returned to pre-stimulation expression levels. Cultured microglia also revealed TRPM4-like, Ca2+-activated non-selective currents (I CAN) with an EC50 of 1.2 microm [Ca2+]i. The expression levels of this current did not change significantly during and after 24 h of LPS exposure. We propose that LPS-induced down-regulation of I IRK and I CRAC will reduce the cell's capacity to produce significant calcium influx upon receptor activation and result in decreased sensitivity to exogenous stimulation. In this scenario, I CAN expression would remain constant, although its activity would automatically be reduced due to the diminished calcium influx capacity of the cell.     

Localization of equilibrative nucleoside transporters in the rat brain

Adenosine is a recognized inhibitory neuromodulator and neuroprotective agent in the central nervous system. It is produced both intra- and extracellularly and transported across the cell membrane. Nucleoside transporters thus have a major impact on the extracellular adenosine levels, and consequently adenosine signalling. We have raised and characterized polyclonal antibodies against both the equilibrative nucleoside transporters 1 and 2, and report for the first time their distribution in rat brain at the cellular level. Double staining studies were performed to assess the localization of the transporters in neural and glial cells. Both transporters were present in practically all neurons. Some astrocytes showed equilibrative nucleoside transporter 1 staining, while equilibrative nucleoside transporter 2 staining on astrocytes was observed only sporadically.     

Chemiluminescence of neutrophil accumulation in liver following ischemia-reperfusion

The median and left lateral lobes of rat liverin situ were rendered ischemic for 30 min, then blood flow re-instituted. After 1, 3, 6, 24, or 48 h livers were removed and set up for isolated perfused organ study. Luminol enhanced chemiluminescence (LEC) was recorded from the surface of the median and left lateral lobe before and for 90 min following phorbol myristate acetate PMA (1.6×10⁻⁸ M) perfusion. An increase in PMA induced luminol enhanced chemiluminescence was evident at 1 h, and continued to increase up to 6 h. By 24 h the magnitude of the PMA response had returned to within control values. This indicates that a large influx of inflammatory cells had occurred in the liver following thein vivo ischemia-reperfusion insult, and that these cells gave a very large and sustained burst of radical production on stimulation with PMA.

     

Glucose enhances mesangial cell apoptosis

Mesangial cell apoptosis occurs in experimental diabetic nephropathy, and this correlates with worsening albuminuria. This study examines the mechanism by which glucose modulates mesangial cell apoptosis. Apoptosis was induced in mesangial cells by serum deprivation in the presence of 5 or 25 mM D-glucose, and examined by expression of Annexin-V and disruption of mitochondrial transmembrane potential. Involvement of Bax, Bcl-2 and NF-kappaB were examined by RT-PCR and EMSA. Involvement of TGF-beta1 was sought by determining the effect of recombinant TGF-beta1on apoptosis and the mediators of the apoptotic pathway (Bcl2/Bax and NF-kappaB). Culture of cells in the presence of 25 mM D-glucose (i) enhanced apoptosis stimulated by serum depletion, (ii) enhanced activation of caspase-3, (iii) inhibited NF-kappaB activation, and (iv) decreased Bcl-2:Bax ratio. Inhibition of NF-kappaB using SN50, also increased mesangial cell apoptosis, and decreased Bcl-2:Bax ratio. Addition of TGF-beta1 to mesangial cells mimicked the effect of high glucose reducing NF-kappaB expression and Bcl-2:Bax ratio. Furthermore glucose-mediated enhanced apoptosis was inhibited by the addition of a blocking antibody to TGF-beta1. Exposure of mesangial cells to 25 mM D-glucose stimulated the generation of both total and active TGF-beta1 in the cell culture supernatant, this increase was only significant after 48-72 h, that is at a time point later than enhanced apoptosis. Addition of 25 mM D-glucose, however, increased sensitivity of mesangial cells to TGF-beta1 as assessed by luciferase activity of a Smad sensitive reporter construct. The data suggest that elevated glucose concentration enhanced the pathway leading to apoptosis following serum deprivation. Furthermore, it is likely that this is dependent on glucose-mediated enhanced sensitivity to endogenous TGF-beta1 rather than glucose stimulated de novo TGF-beta1 synthesis.