Activated Ha-ras induces apoptosis by association with phosphorylated Bcl-2 in a mitogen-activated protein kinase-independent manner.

Serum deprivation of Ha-ras-transformed brown adipocyte cell line resulted in a dramatic apoptotic cell death, as detected either by DNA laddering or by an increase in the percentage of hypodiploid cells or by nuclei condensation and fragmentation, as compared with immortalized cell line or primary fetal brown adipocytes. Moreover, transient transfection of immortalized brown adipocytes with a constitutively active ras gene (Ha-raslys12) mimics the high rate of apoptosis detected in the transformed cell line. On the other hand, transient transfection of the dominant-negative construct of raf-1 rescued serum-deprived Ha-ras-transformed brown adipocytes from apoptosis, decreasing the percentage of hypodiploid cells, the external display of phosphatidylserine, and the DNA laddering. However, inhibition of mitogen-activated protein kinase with PD098059 did not preclude apoptosis and in fact increased the rate of apoptosis observed in serum-deprived Ha-ras-transformed cells, indicating that the Ras/Raf-1 pathway induced apoptosis throughout a mitogen-activated protein kinase kinase 1 (MEK-1)-independent pathway. Furthermore, apoptosis in Ha-ras-transformed brown adipocytes is concurrent with an up-regulation in the expression of the pro-apoptotic protein Bcl-xS, the expression of the anti-apoptotic protein Bcl-2 being down-regulated. Finally, an association of Ras and Raf with phosphorylated Bcl-2 protein was demonstrated in immunoprecipitates from apoptotic cells. Thus, we propose a mechanism of apoptosis in Ha-ras-transformed adipocytes under serum deprivation involving Raf-1 association with phosphorylated Bcl-2, down-regulation of Bcl-2 expression, and up-regulation of Bcl-xS expression.     

Regulation of insulin-like growth factor type I (IGF-I) receptor kinase activity by protein tyrosine phosphatase 1B (PTP-1B) and enhanced IGF-I-mediated suppression of apoptosis and motility in PTP-1B-deficient fibroblasts

The insulin-like growth factor type I (IGF-I) receptor (IGF-IR), activated by its ligands IGF-I and IGF-II, can initiate several signal transduction pathways that mediate suppression of apoptosis, proliferation, differentiation, and transformation. Here we investigated the regulation of IGF-IR activation and function by protein tyrosine phosphatase 1B (PTP-1B). Coexpression of PTP-1B with a beta-chain construct of the IGF-IR (betaWT) inhibited IGF-IR kinase activity in fission yeast Schizosaccharomyces pombe, in COS cells, and in IGF-IR-deficient fibroblasts. In both spontaneously immortalized and simian virus 40 T antigen-transformed embryonic fibroblast cell lines derived from PTP-1B knockout mice, IGF-I induced higher levels of IGF-IR autophosphorylation and kinase activity than were induced in PTP-1B-expressing control cells. PTP-1B-deficient cells exhibited enhanced IGF-I-mediated protection from apoptosis in response to serum withdrawal or etoposide killing, as well as enhanced plating efficiency and IGF-I-mediated motility. Reexpression of PTP-1B in spontaneously immortalized fibroblasts resulted in decreased IGF-IR and AKT activation, as well as decreased protection from apoptosis and decreased motility. These findings demonstrate that PTP-1B can regulate IGF-IR kinase activity and function and that loss of PTP-1B can enhance IGF-I-mediated cell survival, growth, and motility in transformed cells.     

Insulin-induced up-regulated uncoupling protein-1 expression is mediated by insulin receptor substrate 1 through the phosphatidylinositol 3-kinase/Akt signaling pathway in fetal brown adipocytes

To investigate the role of insulin receptor substrate-1 (IRS-1) and its downstream signaling in insulin-induced thermogenic differentiation of brown adipocytes, we have reconstituted IRS-1-deficient fetal brown adipocytes (IRS-1(-/-)) with wild-type IRS-1 (IRS-1(wt)). The lack of IRS-1 resulted in the inability of insulin to induce IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity and Akt phosphorylation in IRS-1(-/-) brown adipocytes. In addition, these cells showed an impairment in activating alpha-Akt, beta-Akt, and gamma-Akt isoforms upon insulin stimulation. Reconstitution of IRS-1(-/-) brown adipocytes with IRS-1(wt) restored the IRS-1/PI 3-kinase/Akt signaling pathway. Treatment of wild-type brown adipocytes with insulin for 24 h up-regulated uncoupling protein-1 (UCP-1) expression and transactivated the UCP-1 promoter; this effect was abolished in the absence of IRS-1 or in the presence of an Akt inhibitor and further recovered after IRS-1(wt) reconstitution. Neither UCP-2 nor UCP-3 was up-regulated by insulin in wild-type and IRS-1-deficient brown adipocytes. Insulin stimulated the expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) and its DNA binding activity in wild-type brown adipocytes but not in IRS-1(-/-) cells. However, insulin stimulation of both C/EBPalpha expression and binding activity was restored after IRS-1(wt) reconstitution of deficient cells. Retrovirus-mediated expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma in IRS-1(-/-) brown adipocytes up-regulated UCP-1 protein content and transactivated UCP-1 promoter regardless of insulin stimulation. Both C/EBPalpha and peroxisome proliferator-activated receptor gamma reconstituted FAS mRNA expression, but only C/EBPalpha restored insulin sensitivity in the absence of IRS-1. Finally, reconstitution of IRS-1(-/-) brown adipocytes with the IRS-1 mutants IRS-1(Phe-895), which lacks IRS-1/growth factor receptor binding protein 2 binding but not IRS-1/p85-PI 3-kinase binding, or with IRS-1(Tyr-608/Tyr-628/Tyr-658), which only binds p85-PI 3-kinase, induced UCP-1 expression and transactivated the UCP-1 promoter. These data provide strong evidence for an essential role of IRS-1 through the PI 3-kinase/Akt signaling pathway inducing UCP-1 gene expression by insulin.     

Susceptibility of brown adipocytes to pro-inflammatory cytokine toxicity and reactive oxygen species

Brown adipose tissue (BAT) cells have a very high oxidative capacity. On the other hand, in obesity and obesity-related diabetes, levels of pro-inflammatory cytokines are elevated, which might promote BAT dysfunction and consequently impair carbohydrate metabolism and thereby exacerbate cellular dysfunction and promote diabetes progression. Therefore, the antioxidative enzyme status of a brown adipocyte cell line and its susceptibility towards pro-inflammatory cytokines, which participate in the pathogenesis of diabetes, and reactive oxygen species (ROS) were analysed. Mature brown adipocytes exhibited significantly higher levels of expression of mitochondrially and peroxisomally located antioxidative enzymes compared with non-differentiated brown adipocytes. Pro-inflammatory cytokines induced a significant decrease in the viability of differentiated brown adipocytes, which was accompanied by a massive ROS production and down-regulation of BAT-specific markers, such as uncoupling protein 1 (UCP-1) and β-Klotho. Taken together, the results strongly indicate that pro-inflammatory cytokines cause brown adipocyte dysfunction and death through suppression of BAT-specific proteins, especially of UCP-1 and β-Klotho, and consequently increased oxidative stress.     

IGF-I receptor activation and BCL-2 overexpression prevent early apoptotic events in human neuroblastoma

The type I insulin-like growth factor receptor (IGF-IR) is important for mitogenesis, transformation, and survival of tumor cells. The current study examines the effect of IGF-IR expression and activation on apoptosis in SHEP human neuroblastoma cells. SHEP cells undergo apoptosis which is prevented by IGF-I addition or overexpression of the IGF-IR (SHEP/IGF-IR cells). High mannitol treatment activates caspase-3 by 1 h in SHEP cells while caspase-3 activation is delayed by 3 h in SHEP/IGF-IR cells. Transfection with Bcl-2 (SHEP/Bcl-2 cells) prevents serum withdrawal and mannitol induced apoptosis and caspase-3 activation. Mannitol induces mitochondrial membrane depolarization in both SHEP and SHEP/IGF-IR cells. IGF-IR activation or overexpression of Bcl-2 in SHEP cells prevents mitochondrial membrane depolarization. Collectively, these results suggest that IGF-IR or Bcl-2 overexpression in neuroblastoma cells promotes cell survival by preventing mitochondrial membrane depolarization and caspase-3 activation, ultimately leading to increased tumor growth.     

Two CD95 (APO-1/Fas) signaling pathways.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).     

IGF-I is a mitogen involved in differentiation-related gene expression in fetal rat brown adipocytes

Fetal rat brown adipocytes at time zero of culture constitute a population of cells of broad spectrum, as estimated by cell size, endogenous fluorescence and lipid content, and show an intrinsic mitogenic competence. They express constitutively early growth-related genes such as c-myc, c-fos, and beta-actin, tissue specific-genes such as the uncoupling protein (UCP) and the lipogenic marker malic enzyme (ME). Fetal brown adipocytes bear a high expression of insulin-like growth factor receptor (IGF-IR), and show a high affinity IGF-I specific-binding to its receptor, and a high number of binding sites per cell. After cell quiescence, insulin-like growth factor I (IGF-I) was as potent as 10% FCS in inducing DNA synthesis, cell number increase, and the entry of cells into the cell-cycle. In addition, IGF- I or 10% FCS for 48 h increased the percentage of [3H]thymidine-labeled nuclei as compared to quiescent cells. Single cell autoradiographic microphotographs show typical multilocular fat droplets brown adipocytes, resulting positive to [3H]thymidine-labeled nuclei in response to IGF-I. IGF-I increased mRNA expression of the early- response genes c-fos (30 min), c-myc (2 and 24 h), and H-ras (4 and 24 h). 10% FCS also increased c-fos and c-myc, but failed to increase H- ras as an early event. IGF-I or 10% FCS, however, similarly increased the mRNA late expression of c-myc, H-ras, c-raf, beta-actin, and glucose 6-phosphate dehydrogenase (G6PD) at 72 h, as compared to quiescent cells. IGF-I or FCS maintained at 24 h or increased at 48 and 72 h UCP mRNA expression. The results demonstrate that IGF-I is a mitogen for fetal rat brown adipocytes, capable of inducing the expression of early and late growth-regulated genes, and of increasing the lipogenic marker ME and the tissue-specific gene UCP, suggesting the involvement of IGF-I in the differentiation as well as in the proliferation processes.     

Noradrenaline induces brown adipocytes cell growth via beta-receptors by a mechanism dependent on ERKs but independent of cAMP and PKA

It has been well established that the key role of noradrenaline is the induction of uncoupling-protein-1 (UCP-1) expression, the unique marker of brown adipocytes. However, its implication on proliferation and the pathways involved are not as well characterized. By using rat fetal brown adipocytes as a model, we show that, although noradrenaline activates extracellular regulated kinases (ERKs) through beta-, alpha1-, and alpha2-receptors, only beta-receptors mediate cell growth by a mechanism that requires ERKs activation but is independent of cyclic-adenosine-monophosphate/protein kinase A (cAMP/PKA). Conversely, the cAMP/PKA cascade mediates noradrenaline-induced UCP-1 expression, whereas ERKs pathway attenuates thermogenic differentiation. On the other hand, alpha1- and alpha2-receptors have an antiproliferative effect that is enhanced by ERK inhibition.     

Effect of the beta(3)-adrenergic agonist Cl316,243 on functional differentiation of white and brown adipocytes in primary cell culture

We investigated the effect of the specific beta(3)-adrenergic receptor agonist CL 316,243 (CL) on proliferation and functional differentiation of the Siberian hamster (Phodopus sungorus) white and brown preadipocytes in primary cell culture. Proliferation of both white and brown preadipocytes was stimulated by a general beta-adrenergic agonist (isoproterenol) but not by CL. Lipolysis of differentiated white and brown adipocytes was stimulated similarly by CL with maximum effect at 10 nM. Thermogenic properties of cells were assessed by immunodetection of UCP-1, the brown adipocyte specific uncoupling protein, and measurement of cytochrome c oxidase (COx) activity as an index of mitochondrial capacity. UCP-1 content was largely increased by CL in BAT but not in WAT cultures. Basal UCP-2 mRNA levels were similar in WAT and BAT cultures and increased by both CL and isoproterenol. COx activity of BAT cultures was twice as high as that of WAT cultures but in neither cell culture system could it be increased by beta-adrenergic stimulation. We suggest (i) that white and brown preadipocyte proliferation is increased in vitro via beta1 or beta(2), but not beta(3)-adrenergic pathways, (ii) that white and brown preadipocytes represent different cell types, and (iii) that in vitro beta-adrenergic stimulation it is not sufficient to induce complete thermogenic adaptation of brown adipocytes.     

Inhibition of Caspases Rescues Brown Adipocytes from Apoptosis Downregulating BCL-XS and Upregulating BCL-2 Gene Expression

Serum deprivation of the immortalized brown adipocyte cell line resulted in growth arrest inG₀/G₁phases of the cell cycle and apoptosis, as detected by DNA laddering, nuclei condensation and fragmentation, and an increase in the percentage of hypodiploid cells. In addition, apoptosis in these cells is accompanied by an induction of the expression of the apoptotic form of the Bcl-x gene, the isoform Bcl-xS, and by a decrease of Bcl-2 expression, Bcl-xL remaining almost undetectable. The loss of mitochondrial membrane potential was associated with apoptosis. Z-VAD, a cell-permeable inhibitor of caspases, but not cycloheximide, precludes DNA laddering under serum deprivation. Moreover, Z-VAD rescues serum-deprived brown adipocytes from apoptosis, decreasing the percentage of hypodiploid cells, the percentage of apoptotic cells under Tunnel assay, and the external display of phosphatidylserine. More importantly, Z-VAD survival effects on immortalized brown adipocytes concur with a downregulation of Bcl-xS mRNA/protein and an upregulation of Bcl-2 protein content. Ultimately, Z-VAD prevents the loss of mitochondrial membrane potential.     

Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes

Murine 3T3-L1 preadipocytes proliferate normally in medium containing fetal calf serum depleted of insulin, growth hormone, and insulin-like growth factor-I (IGF-I). However, the cells do not differentiate into adipocytes in the presence of the hormone-depleted serum. Supplementation of the growth medium with 10-20 nM IGF-I or 2 microM insulin restores the ability of 3T3-L1 cells to develop into adipocytes. The cells acquire an adipocyte morphology, accumulate triglycerides, and express a 450-fold increase in the activity of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. The increase in glycerol-3-phosphate dehydrogenase activity is paralleled by the accumulation of glycerol-3-phosphate dehydrogenase mRNA and mRNA for the myelin P2-like protein aP2, another marker for fat cell development. IGF-I or insulin-stimulated adipogenesis in 3T3-L1 cells is not dependent on growth hormone. Occupancy of preadipocyte IGF-I receptors by IGF-I (or insulin) is implicated as a central step in the differentiation process. The IGF-I receptor binds insulin with a 70-fold lower affinity than IGF-I, and 30-70-fold higher levels of insulin are required to duplicate the effects of an optimal amount of IGF-I. The effects of 10-20 nM IGF-I are likely to be mediated by high affinity (KD = 5 nM) IGF-I receptors that are expressed at a density of 13,000 sites/preadipocyte. In undifferentiated cells the IGF-I receptor concentration is twice that of the insulin receptor. After adipocyte differentiation is triggered, the number and affinity of IGF-I receptors remain constant while insulin receptor number increases approximately 25-fold as developing adipocytes become responsive to insulin at the level of metabolic regulation. Thus, preadipocytes have the potential for a maximal response to IGF-I, whereas the accumulation of more than 95% of adipocyte insulin receptors and the appearance of responsiveness to insulin are consequences of differentiation. IGF-I or insulin is essential for the induction of a variety of abundant and nonabundant mRNAs characteristic of 3T3-L1 adipocytes.     

Synergistic activation of UCP-3 expression in cultured fetal rat brown adipocytes by PPARalpha and PPARgamma ligands

Rat brown adipocytes express mRNAs for Uncoupling Proteins (UCP) 1, 2 and 3 and the Peroxisome Proliferator Activated Receptors (PPAR) alpha and gamma. We have examined the effects of selective PPARalpha or -gamma activation on changes in UCP-1 and UCP-3 mRNA levels in cultured fetal rat brown adipocytes (FBA). Rosiglitazone (1.0 microM), a selective PPARgamma agonist, elicited 5- and 3-fold increases in UCP-1 and UCP-3, respectively. The PPARalpha ligand, Wy14643 (10.0 microM) increased UCP-3 tenfold, but decreased UCP-1. A synergistic effect on UCP-3 expression (30-fold increase; P < 0. 05) was observed when FBA were exposed to a combination of Wy14643 (10.0 microM) and rosiglitazone (10.0 microM). Thus, activation of PPARgamma increases UCP-1 and UCP-3 levels which are differentially regulated by PPARalpha. A synergistic interaction occurs between PPARalpha and PPARgamma in the regulation of UCP-3 in FBA, probably via co-activator recruitment, suppression of co-repressor proteins or through a direct interaction at the level of the PPRE.     

Novel adipocyte lines from brown fat: a model system for the study of differentiation, energy metabolism, and insulin action

Adipose tissue has emerged as an important endocrine regulator of glucose metabolism and energy homeostasis. By virtue of the mitochondrial protein uncoupling protein-1 (UCP-1), brown fat additionally plays a unique role in thermoregulation. Interest has focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Most studies of adipocytes have been limited either to primary cell culture or to a small number of established cell lines. Recently, we have generated immortalized brown adipocyte cell lines from single newborn mice of different knockout mouse models. These cell lines retain the main characteristics of primary cells including UCP-1 expression. They display sensitive and diverse metabolic responses to insulin and adrenergic stimulation and have proven to be useful in the characterization of UCP regulation and the role of key insulin signaling elements for insulin action. Here, we outline common approaches to the generation of adipose tissue cell lines. Furthermore, we propose that the novel technique of generating brown adipocyte lines from a single newborn mouse will be instrumental in gaining further insight into the role of a broad range of signaling molecules in adipose tissue biology and in the pathogenesis of insulin resistance.     

Unknotting the roles of Bcl-2 and Bcl-xL in cell death

The antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL play important roles in inhibiting mitochondria-dependent extrinsic and intrinsic cell death pathways. It seems that these two proteins have distinct functions for inhibiting extrinsic and intrinsic cell death pathways. The overexpression of Bcl-2 is able to inhibit not only apoptotic cell death but also in part nonapoptotic cell death, which has the role of cell cycle arrest in the G1 phase, which may promote cellular senescence. The overexpression of Bcl-2 may also have the ability to enhance cell death in the interaction of Bcl-xL with other factors. The overexpression of Bcl-xL enhances autophagic cell death when apoptotic cell death is inhibited in Bax(-/-)/Bak(-/-) double knockout cells. This review discusses the previously unexplained aspects of Bcl-2 and Bcl-xL functions associated with cell death, for better understanding of their functions in the regulation.     

Quercetin regulates insulin like growth factor signaling and induces intrinsic and extrinsic pathway mediated apoptosis in androgen independent prostate cancer cells (PC-3)

Progression of prostate cancer is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. To investigate the effect of quercetin on insulin-like growth factor signaling and apoptosis in androgen independent prostate cancer cells (PC-3), IGF-IR, PI-3K, p-Akt, Akt, cyclin D1, Bad, cytochrome c, PARP, caspases-9 and 10 protein levels were assessed by western blot analysis. Mitochondrial membrane potency was detected by rhodamine-123 staining. Quercetin induced caspase-3 activity assay was performed for activation of apoptosis. Further, RT-PCR was also performed for Bad, IGF-I, II, IR, and IGFBP-3 mRNA expression. Quercetin significantly increases the proapoptotic mRNA levels of Bad, IGFBP-3 and protein levels of Bad, cytochrome C, cleaved caspase-9, caspase-10, cleaved PARP and caspase-3 activity in PC-3 cells. IGF-IRβ, PI3K, p-Akt, and cyclin D1 protein expression and mRNA levels of IGF-I, II and IGF-IR were decreased significantly. Further, treatment with PI3K inhibitor (LY294002) and quercetin showed decreased p-Akt levels. Apoptosis is confirmed by loss of mitochondrial membrane potential in quercetin treated PC-3 cells. This study suggests that quercetin decreases the survival of androgen independent prostate cancer cells by modulating the expression of insulin-like growth factors (IGF) system components, signaling molecules and induces apoptosis, which could be very useful for the androgen independent prostate cancer treatment.     

Deletion of the BH1 domain of Bcl-2 accelerates apoptosis by acting in a dominant negative fashion

To investigate the exact biochemical functions by which Bcl-2 regulates apoptosis, we established a stable human small cell lung carcinoma cell line, Ms-1, overexpressing wild-type human Bcl-2 or various deletion and point mutants thereof, and examined the effect of these Bcl-2 mutants on apoptosis induced by antitumor drugs such as camptothecin. Cytochrome c release, caspase-3-(-like) protease activation, and apoptosis induced by antitumor drugs were accelerated by overexpression of Bcl-2 lacking a Bcl-2 homology (BH) 1 domain (Bcl-2/ DeltaBH1), but not by that of BH2, BH3, or BH4 domain-deleted Bcl-2. A similar result was obtained upon the substitution of glycine 145 with alanine in the BH1 domain (Bcl-2/G145A), which failed to interact with either Bax or Bak. Pro-apoptotic Bax and Bak have been known to be activated in response to antitumor drugs, and Bcl-2/G145A as well as Bcl-2/DeltaBH1 also accelerated Bax- or Bak-induced apoptosis in HEK293T cells. These two mutants still retained the ability to interact with wild-type Bcl-2 and Bcl-xL, and abrogated the inhibitory effect of wild-type Bcl-2 or Bcl-xL on Bax- or Bak-induced apoptosis. In addition, immunoprecipitation studies revealed that Bcl-2/DeltaBH1 and Bcl-2/G145A interrupted the association between wild-type Bcl-2 and Bax/Bak. Taken together, our results demonstrate that Bcl-2/DeltaBH1 or Bcl-2/G145A acts as a dominant negative of endogenous anti-apoptotic proteins such as Bcl-2 and Bcl-xL, thereby enhancing antitumor drug-induced apoptosis, and that this dominant negative activity requires both a failure of interaction with Bax and Bak through the BH1 domain of Bcl-2 and retention of the ability to interact with Bcl-2 and Bcl-xL.     

The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.