CpG-Oligodeoxynucleotide Treatment Protects against Ionizing Radiation-Induced Intestine Injury

Background

the bone marrow and the intestine are the major sites of ionizing radiation (IR)-induced injury. Our previous study demonstrated that CpG-oligodeoxynucleotide (ODN) treatment mitigated IR-induced bone marrow injury, but its effect on the intestine is not known. In this study, we sought to determine if CpG-ODN have protective effect on IR-induced intestine injury, and if so, to determine the mechanism of its effect.

Methods and Findings

Mice were treated with CpG-ODN after IR. The body weight and survival were daily monitored for 30 days consecutively after exposure. The number of surviving intestinal crypt was assessed by the microcolony survival assay. The number and the distribution of proliferating cell in crypt were evaluated by TUNEL assay and BrdU assay. The expression of Bcl-2, Bax and caspase-3 in crypt were analyzed by Immunohistochemistry assay. The findings showed that the treatment for irradiated mice with CpG-ODN diminished body weight loss, improved 30 days survival, enhanced intestinal crypts survival and maintained proliferating cell population and regeneration in crypt. The reason might involve that CpG-ODN up-regulated the expression of Bcl-2 protein and down-regulated the expression of Bax protein and caspase-3 protein.

Conclusion

CpG-ODN was effective in protection of IR-induced intestine injury by enhancing intestinal crypts survival and maintaining proliferating cell population and regeneration in crypt. The mechanism might be that CpG-ODN inhibits proliferating cell apoptosis through regulating the expression of apoptosis-related protein, such as Bax, Bcl-2 and caspase-3.     

Brain-derived neurotrophic factor prevents changes in Bcl-2 family members and caspase-3 activation induced by excitotoxicity in the striatum

Brain-derived neurotrophic factor (BDNF) prevents the loss of striatal neurons caused by excitotoxicity. We examined whether these neuroprotective effects are mediated by changes in the regulation of Bcl-2 family members. We first analyzed the involvement of the phosphatidylinositol 3-kinase/Akt pathway in this regulation, showing a reduction in phosphorylated Akt (p-Akt) levels after both quinolinate (QUIN, an NMDA receptor agonist) and kainate (KA, a non-NMDA receptor agonist) intrastriatal injection. Our results also show that Bcl-2, Bcl-x(L) and Bax protein levels and heterodimerization are selectively regulated by NMDA and non-NMDA receptor stimulation. Striatal cell death induced by QUIN is mediated by an increase in Bax and a decrease in Bcl-2 protein levels, leading to reduced levels of Bax:Bcl-2 heterodimers. In contrast, changes in Bax protein levels are not required for KA-induced apoptotic cell death, but decreased levels of both Bax:Bcl-2 and Bax:Bcl-x(L) heterodimer levels are necessary. Furthermore, QUIN and KA injection activated caspase-3. Intrastriatal grafting of a BDNF-secreting cell line counter-regulated p-AKT, Bcl-2, Bcl-x(L) and Bax protein levels, prevented changes in the heterodimerization between Bax and pro-survival proteins, and blocked caspase-3 activation induced by excitotoxicity. These results provide a possible mechanism to explain the anti-apoptotic effect of BDNF against to excitotoxicity in the striatum through the regulation of Bcl-2 family members, which is probably mediated by Akt activation.     

Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c.

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.     

Apoptosis of Mo7e leukemia cells is associated with the cleavage of Bcl-2 into a shortened fragment that is not functional for heterodimerization with Bcl-2 and Bax

Bcl-2 is an integral intracellular membrane protein that can protect cells from apoptosis induced by multiple insults in a variety of cell types. During apoptosis, Bcl-2 was cleaved into a shortened fragment (Bcl-2/Delta34) by a caspase-3-like protease in human Mo7e megakaryocytic leukemia cells deprived of exogenous rhGM-CSF. Results from cell fractionation and immunoblot analyses indicated that both Bcl-2 and Bcl-2/Delta34 were located exclusively on the mitochondria of Mo7e cells. Treatment of isolated mitochondria with recombinant caspase-3 induced the same cleavage of Bcl-2 in vitro and caused the release of cytochrome c from the mitochondria into the supernatant. The antiapoptotic effect of Bcl-2/Delta34 was investigated using an in vitro protein translation approach. Both Bcl-2/Delta34 and Bax proteins generated in wheat germ extract were readily relocated to the mitochondria isolated from control Mo7e cells. Insertion of Bax, but not Bcl-2/Delta34, into mitochondria triggered a rapid release of cytochrome c from the mitochondria. Coimmunoprecipitation studies showed that, unlike Bcl-2, the cleaved Bcl-2 fragment was no longer functional for dimerization with either Bcl-2 or Bax. Taken together, these findings showed that the integrity of Bcl-2 is necessary for its function of heterodimerization with Bax, which appears to be one of the mechanisms of antiapoptotic effect of Bcl-2.     

NH2-terminal BH4 domain of Bcl-2 is functional for heterodimerization with Bax and inhibition of apoptosis.

The Bcl-2 family proteins comprise pro-apoptotic as well as anti-apoptotic members. Heterodimerization between members of the Bcl-2 family proteins is a key event in the regulation of apoptosis. We report here that Bcl-2 protein was selectively cleaved by active caspase-3-like proteases in CTLL-2 cell apoptosis in response to interleukin-2 deprivation. Structural and functional analyses of the cleaved fragment revealed that the NH2-terminal region of Bcl-2 (1-34 amid acids) was required for its anti-apoptotic activity and heterodimerization with pro-apoptotic Bax protein. Site-directed mutagenesis of the NH2-terminal region showed that substitutions of hydrophobic residues of BH4 domain resulted in the loss of ability to form a heterodimer with Bax. Particularly instructive was that the V15E mutant of Bcl-2, which completely lost the ability to form a heterodimer with Bax, failed to inhibit Bax- and staurosporine-induced apoptosis. Our results suggest that the BH4 domain of Bcl-2 is critical for its heterodimerization with Bax and for exhibiting anti-apoptotic activity. Therefore, agents interferring with the critical residues of the BH4 domain may provide a new strategy in cancer therapy by impairing Bcl-2 function.     

Modulation of apoptosis of eckol against ionizing radiation in mice

To investigate the radioprotective potential of eckol, a component of the seaweed Ecklonia cava, against radiation in vivo, we evaluated the effect of eckol on cyto- and histo-protective capability of the lymphocytes and intestine against damage induced by a single whole body irradiation (WBI) in vivo. Here, we ascertained that eckol protected the lymphocytes’ viability and rescued intestinal cells from radiation-induced apoptosis by decreasing the amount of pro-apoptotic p53 and Bax and increasing that of anti-apoptotic Bcl-2. These findings indicate that the overexpression of anti-apoptotic protein, which may lead to resistance to DNA damage, is involved deeply in protection of gastrointestinal cells after irradiation. Thus, eckol that can protect cells and tissues against ionizing radiation may have considerable potential as adjuncts to successful radiotherapy.     

Synthesis and radioprotective effects of novel hybrid compounds containing edaravone analogue and 3-n-butylphthalide ring-opening derivatives

As the potential risk of radiation exposure is increasing, radioprotectors studies are gaining importance. In this study, novel hybrid compounds containing edaravone analogue and 3-n-butylphthalide ring-opening derivatives were synthesized, and their radioprotective effects were evaluated. Among these, compound 10a displayed the highest radioprotective activity in IEC-6 and HFL-1 cells. Its oral administration increased the survival rates of irradiated mice and alleviated total body irradiation (TBI)-induced hematopoietic damage by mitigating myelosuppression and improving hematopoietic stem/progenitor cell frequencies. Furthermore, 10a treatment prevented abdominal irradiation (ABI)-induced structural damage to the small intestine. Experiment results demonstrated that 10a increased the number of Lgr5⁺ intestinal stem cells, lysozyme⁺ Paneth cells and Ki67⁺ transient amplifying cells, and reduced apoptosis of the intestinal epithelium cells in irradiated mice. Moreover, in vitro and in vivo studies demonstrated that the radioprotective activity of 10a is associated to the reduction of oxidative stress and the inhibition of DNA damage. Furthermore, compound 10a downregulated the expressions of p53, Bax, caspase-9 and caspase-3, and upregulated the expression of Bcl-2, suggesting that it could prevent irradiation-induced intestinal damage through the p53-dependent apoptotic pathway. Collectively, these findings demonstrate that 10a is beneficial for the prevention of radiation damage and has the potential to be a radioprotector.     

Ionizing Radiation Potentiates Dihydroartemisinin-Induced Apoptosis of A549 Cells via a Caspase-8-Dependent Pathway

This report is designed to explore the molecular mechanism by which dihydroartemisinin (DHA) and ionizing radiation (IR) induce apoptosis in human lung adenocarcinoma A549 cells. DHA treatment induced a concentration- and time-dependent reactive oxygen species (ROS)-mediated cell death with typical apoptotic characteristics such as breakdown of mitochondrial membrane potential (Δψ_m), caspases activation, DNA fragmentation and phosphatidylserine (PS) externalization. Inhibition of caspase-8 or -9 significantly blocked DHA-induced decrease of cell viability and activation of caspase-3, suggesting the dominant roles of caspase-8 and -9 in DHA-induced apoptosis. Silencing of proapoptotic protein Bax but not Bak significantly inhibited DHA-induced apoptosis in which Bax but not Bak was activated. In contrast to DHA treatment, low-dose (2 or 4 Gy) IR induced a long-playing generation of ROS. Interestingly, IR treatment for 24 h induced G2/M cell cycle arrest that disappeared at 36 h after treatment. More importantly, IR synergistically potentiated DHA-induced generation of ROS, activation of caspase-8 and -3, irreparable G₂/M arrest and apoptosis, but did not enhance DHA-induced loss of Δψ_m and activation of caspase-9. Taken together, our results strongly demonstrate the remarkable synergistic efficacy of combination treatment with DHA and low-dose IR for A549 cells in which IR potentiates DHA-induced apoptosis largely by enhancing the caspase-8-mediated extrinsic pathway.     

Ionizing radiation can overcome resistance to TRAIL in TRAIL-resistant cancer cells

Although the majority of cancer cells are killed by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand treatment), certain types show resistance to it. Ionizing radiation also induces cell death in cancer cells and may share common intracellular pathways with TRAIL leading to apoptosis. In the present study, we examined whether ionizing radiation could overcome TRAIL resistance in the variant Jurkat clones. We first selected TRAIL-resistant or -sensitive Jurkat clones and examined cross-responsiveness of the clones between TRAIL and radiation. Treatment with gamma-radiation induced significant apoptosis in all the clones, indicating that there seemed to be no cross-resistance between TRAIL and radiation. Combined treatment of radiation with TRAIL synergistically enhanced killing of TRAIL-resistant cells, compared to TRAIL or radiation alone. Apoptosis induced by combined treatment of TRAIL and radiation in TRAIL-resistant cells was associated with cleavage of caspase-8 and the proapoptotic Bid protein, resulting in the activation of caspase-9 and caspase-3. No changes in the expressions of TRAIL receptors (DR4 and DR5) and Bcl-2 or Bax were found after treatment. The caspase inhibitor z-VAD-fmk completely counteracted the synergistic cell killing induced by combined treatment of TRAIL and gamma-radiation. These results demonstrated that ionizing radiation in combination with TRAIL could overcome resistance to TRAIL in TRAIL-resistant cells through TRAIL receptor-independent synergistic activation of the cascades of the caspase-8 pathway, suggesting a potential clinical application of combination treatment of TRAIL and ionizing radiation to TRAIL-resistant cancer cells.     

Phloroglucinol Protects Small Intestines of Mice from Ionizing Radiation by Regulating Apoptosis-Related Molecules: A Comparative Immunohistochemical Study

Phloroglucinol (PG) is a phenolic compound isolated from Ecklonia cava, a brown algae abundant on Jeju island, Korea. Previous reports have suggested that PG exerts antioxidative and cytoprotective effects against oxidative stress. In this study, we confirmed that PG protected against small intestinal damage caused by ionizing radiation, and we investigated its protective mechanism in detail. Regeneration of intestinal crypts in the PG-treated irradiated group was significantly promoted compared with that in irradiated controls. The expression level of proapoptotic molecules such as p53, Bax, and Bak in the small intestine was downregulated and that of antiapoptotic molecules such as Bcl-2 and Bcl-X_S/L was augmented in the PG-treated group. On histological observation of the small intestine, PG inhibited the immunoreactivity of p53, Bax, and Bak and increased that of Bcl-2 and Bcl-X_S/L. These results demonstrate the protective mechanisms of PG in mice against intestinal damage from ionizing radiation, providing the benefit of raising the apoptosis threshold of jejunal crypt cells.     

MicroRNA-216b inhibits heat stress-induced cell apoptosis by targeting Fas in bovine mammary epithelial cells

Heat stress affects milk yield and quality in lactating dairy cows in summer. Bovine mammary epithelial cells (bMECs) play a key role in milk secretion, and microRNAs (miRNAs) regulate numerous functions of bMEC. Previous reports have verified that miR-216b regulated cell apoptosis through repressing target genes in several cancer cells. So, our purpose was to explore the potential involvement of miR-216b in heat stress-induced cell apoptosis in bMECs. Firstly, the heat stress model was constructed and we found that apoptotic rates of bMECs significantly increased under heat stress. The expression of miR-216b, Bax mRNA, and caspase-3 mRNA was upregulated. However, Bcl-2 mRNA level was detected to differentially downregulated. Overexpression of miR-216b remarkably downregulated the expression of caspase-3 and Bax mRNA and protein, and the mRNA and protein level of Bcl-2 was increased. Inhibition of miR-216b increased the activity of caspase-3 and Bax, and the level of Bcl-2 was inhibited. Moreover, Fas was identified as a target gene of miR-216b through bioinformatic analysis and dual-luciferase reporter assay. Fas activity was significantly inhibited and enhanced respectively after transfecting miRNA mimics and inhibitor. Finally, inhibition of Fas via the small interfering RNA (siRNA) also inhibited cell apoptosis induced by heat stress. Taken together, our results indicated that miR-216b exerted as an anti-apoptotic effect under heat stress in bMECs by targeting Fas.     

Concomitant Inhibition of MDM2 and Bcl-2 Protein Function Synergistically Induce Mitochondrial Apoptosis in AML

Disruption of Mdm2-p53 interaction activates p53 signaling, disrupts the balance ofantiapoptotic and proapoptotic Bcl-2 family proteins and induces apoptosis in acutemyeloid leukemia (AML). Overexpression of Bcl-2 may inhibit this effect. Thus,functional inactivation of antiapoptotic Bcl-2 proteins may enhance apoptogenic effects ofMdm2 inhibition. We here investigate the potential therapeutic utility of combinedtargeting of Mdm2 by Nutlin-3a and Bcl-2 by ABT-737, recently developed inhibitors ofprotein-protein interactions. Nutlin-3a and ABT-737 induced Bax conformational changeand mitochondrial apoptosis in AML cells in a strikingly synergistic fashion. Nutlin-3ainduced p53-mediated apoptosis predominantly in S and G₂/M cells, while cells in G₁ were protected through induction of p21. In contrast, ABT-737 induced apoptosis predominantly in G₁ , the cell cycle phase with the lowest Bcl-2 protein levels and Bcl-2/Bax ratios. In addition, Bcl-2 phosphorylation on Ser⁷⁰ was absent in G₁ but detectable in G₂/M, thus lower Bcl-2 levels and absence of Bcl-2 phosphorylation appeared to facilitate ABT-737-induced apoptosis of G₁ cells. The complementary effects of Nutlin-3a and ABT-737 in different cell cycle phases could, in part, account for their synergistic activity. Our data suggest that combined targeting of Mdm2 and Bcl-2 proteins could offer considerable therapeutic promise in AML.     

Apoptosis modulatory activities of transiently expressed Bcl-2: Roles in cytochrome c release and Bax regulation

Bcl-2 and Bcl-X_L are pro-survival members of the Bcl-2 family. These proteins have been shown to antagonize the pro-apoptotic activity of Bax and promote cell survival through blocking Bax translocation from the cytosol to mitochondria and by preventing the release of cytochrome c. However, it has been recently reported that transiently expressed Bcl-2 unexpectedly leads to significant cell toxicity. To study this intriguing phenomenon, we have carried out further analyses into the properties of transiently expressed Bcl-2. We found that various isoforms of human and different species of Bcl-2 were equally capable of inducing apoptosis. In addition, we discovered that transient expression of Bcl-2, unlike its pro-survival homolog Bcl-X_L, can lead to the release of cytochrome c from mitochondria and that the resulting cell death can be inhibited by caspase and calpain inhibitors. Moreover, we have shown that unlike the pro-apoptotic protein Bid, the toxicity associated with the transient expression of Bcl-2 occurs independent of the activity of the endogenous Bax. Finally, we found that in spite of its intrinsic toxicity, transiently expressed Bcl-2 is fully capable of blocking the ectopically expressed Bax from localizing to mitochondria. Taken together, these studies demonstrate that transiently expressed Bcl-2 displays opposing functional properties.     

Evaluation of Insulin and Ascorbic Acid Effects on Expression of Bcl-2 Family Proteins and Caspase-3 Activity in Hippocampus of STZ-Induced Diabetic Rats

Aims Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic rats were evaluated in this study. Methods Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of Bcl-2, Bcl-x_L, and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2, Bcl-x_L, and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method. Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit. Results Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-x_L proteins expression. The Bax/Bcl-2 and Bax/Bcl-x_L ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted in decrease in Bax protein expression and increase in Bcl-2 and Bcl-x_L proteins expression. The Bcl-2/Bax and Bcl-x_L/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity. Conclusions Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus of STZ-induced diabetic rats.     

Bax κ, a novel Bax splice variant from ischemic rat brain lacking an ART domain, promotes neuronal cell death

Bax is a pro-apoptotic Bcl-2 family protein that regulates programmed cell death through homodimerization and through heterodimerization with Bcl-2. Bax alpha is encoded by six exons and undergoes alternative splicing. Bax kappa, a splice variant of Bax with conserved BH1, BH2 and BH3 binding domains and a C-terminal transmembrane domain (TM), but with an extra 446-bp insert between exons 1 and 2 leading to loss of an N-terminal ART domain, was identified from an ischemic rat brain cDNA library. Expression of Bax kappa mRNA and protein was up-regulated in hippocampus after cerebral ischemic injury. The increased Bax kappa mRNA was distributed mainly in selectively vulnerable hippocampal CA1 neurons that are destined to die after global ischemia. Overexpression of Bax kappa protein in HN33 mouse hippocampal neuronal cells induced cell death, which was partially abrogated by co-overexpression of Bcl-2. Moreover, co-overexpression of Bax kappa and Bax alpha increased HN33 cell death. The results suggest that the Bax kappa may have a role in ischemic neuronal death.     

Neuroprotectin D1 Induces Dephosphorylation of Bcl-xL in a PP2A-dependent Manner during Oxidative Stress and Promotes Retinal Pigment Epithelial Cell Survival

Retinal pigment epithelial (RPE) cell integrity is critical for the survival of photoreceptor cells. Bcl-x_L is a major anti-apoptotic Bcl-2 protein required for RPE cell survival, and phosphorylation of Bcl-x_L at residue Ser-62 renders this protein pro-apoptotic. In this study, we identify serine/threonine protein phosphatase 2A (PP2A) as a key regulator of Bcl-x_L phosphorylation at residue Ser-62 in ARPE-19 cells, a spontaneously arising RPE cell line in which Bcl-x_L is highly expressed. We found that either PP2A inhibitor okadaic acid or depletion of catalytic subunit α of PP2A (PP2A/Cα) by small interfering RNA enhanced Bcl-x_L phosphorylation when activated with hydrogen peroxide and tumor necrosis factor α-induced oxidative stress. Disruption of PP2A/Cα exacerbated oxidative stress-induced apoptosis. PP2A/Cα colocalized and interacted with S62Bcl-x_L in cells stressed with H₂O₂/tumor necrosis factor α. By contrast, the omega-3 fatty acid docosahexaenoic acid derivative, neuroprotectin D1 (NPD1), a potent activator of survival signaling, down-regulated oxidative stress-induced phosphorylation of Bcl-x_L by increasing protein phosphatase activity. NPD1 also attenuated the oxidative stress-induced apoptosis by knockdown of PP2A/Cα and increased the association of PP2A/Cα with S62Bcl-x_L as well as total Bcl-x_L. NPD1 also enhanced the heterodimerization of Bcl-x_L with its counterpart, pro-apoptotic protein Bax. Thus, NPD1 modulates the activation of this Bcl-2 family protein by dephosphorylating in a PP2A-dependent manner, suggesting a coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress.     

Peptides derived from BH3 domains of Bcl-2 family members: a comparative analysis of inhibition of Bcl-2, Bcl-x(L) and Bax oligomerization,induction of cytochrome c release,and activation of cell death

Overexpression of Bcl-2, an anti-apoptotic oncoprotein, is commonly observed in a variety of human malignancies and is associated with resistance to chemotherapy and radiotherapy. Although the precise mechanism of Bcl-2 action remains elusive, current evidence indicates that Bcl-2 inhibits apoptosis by binding and inhibiting pro-apoptotic molecules such as Bax. Therefore, agents that disrupt the ability of Bcl-2, or other anti-apoptotic molecules, to bind to pro-apoptotic molecules may have therapeutic value. Several studies have shown that the BH3 domains of Bcl-2 and Bax are critically important for Bax/Bcl-2 heterodimerization. In this report, we designed and synthesized peptides based on the BH3 domains of three distinct Bcl-2 family members, Bcl-2, Bax and Bad. In vitro interaction assays were used to compare the abilities of the different peptides to inhibit Bax/Bcl-2 and Bax/Bcl-x(L) heterodimerization, as well as Bcl-2 and Bax homodimerization. Bax BH3 peptide (20-amino acids) potently inhibited both Bax/Bcl-2 and Bax/Bcl-x(L) interactions, exhibiting IC(50) values of 15 and 9.5 microM, respectively. The Bad BH3 peptide (21 amino acids) was slightly more potent than Bax BH3 at inhibiting Bax/Bcl-x(L) but failed to disrupt Bax/Bcl-2. Bcl-2 BH3 peptide (20-amino acids) was inactive toward Bax/Bcl-2 and had only a weak inhibitory effect on Bax/Bcl-x(L) heterodimerization. All three BH3 peptides failed to significantly inhibit homodimerization of Bcl-2 or Bax. Consistent with its ability to disrupt Bax/Bcl-2 heterodimerization, Bax BH3 peptide was able to overcome Bcl-2 overexpression and induce cytochrome c release from mitochondria of Bcl-2-overexpressing Jurkat T leukemic cells. Bad BH3 peptide, while potently inducing cytochrome c release in wild-type Jurkat cells, only partially overcame the effects of Bcl-2 overexpression. Bcl-2 BH3 failed to induce cytochrome c release, even in wild-type cells. Delivery of the Bax BH3 and Bad BH3 peptides into wild-type Jurkat cells induced comparable levels of cell death. In cells overexpressing Bcl-2, the potency of Bax BH3 peptide was similar to that seen in wild-type cells, while the efficacy of Bad BH3 peptide was reduced. By contrast, in Bcl-x(L)-overexpressing cells, Bad BH3 exhibited greater cell-killing activity than Bax BH3. The Bcl-2 BH3 peptide and a mutant Bax BH3 peptide had no appreciable effect on Jurkat cells. Together, our data suggest that agents based on the Bax BH3 domain may have therapeutic value in cancers overexpressing Bcl-2, while agents based on the BH3 domain of Bad may be more useful for tumors overexpressing Bcl-x(L).     

The age-dependent induction of apoptosis-inducing factor (AIF) in the human semitendinosus skeletal muscle

To assess the dependence on age of the expression of apoptosis regulatory proteins in the human semitendinosus muscle, we measured the expression levels of several apoptosis-related genes, including apoptosis-inducing factor (AIF), Bax, Bcl-2, caspase-3 and heat shock protein 70 (HSP70), using RT-PCR, immunohistochemistry and TUNEL assays. We found that the DNA fragmentation was proportional to the age of the tissues sample donors. The expression levels of AIF were significantly elevated (by 10 to 25%) in semitendinosus tissue samples from older individuals, but the Bax, Bcl-2, caspase-3 and HSP 70 levels remained almost constant. This data suggests that the morphological and functional changes observed in aged human semitendinosus muscle correlates with the apoptosis of muscle cells through the induction of AIF.