Biomedical subjects

G Nuñez

Publications and source records attributed to G Nuñez.

At least 19 recordsLinked to original sources

PubMed1998

Caspases: the proteases of the apoptotic pathway.

Apoptosis, a morphologically defined form of physiological cell death, is implemented by a death machinery whose executionary arm is a family of cysteine proteases called caspases. These death proteases are part of a proteolytic caspase cascade that is activated by diverse apoptotic stimuli from outside and inside of the cell. The cell death machinery is evolutionarily conserved and composed of caspases and their regulatory components that include activators and repressors. These key components of the death machinery are linked to signaling pathways that are activated by either ligation of death receptors expressed at the cell surface or intracellular death signals. Caspases are normally present in the cell as proenzymes that require limited proteolysis for activation of enzymatic activity. Recent studies suggest that the basic mechanism of caspase activation is conserved in evolution. Binding of initiator caspase precursors to activator molecules appears to promote procaspase oligomerization and autoactivation. Enzymatic activation of initiator caspases leads to proteolytic activation of downstream (effector) caspases and cleavage of a number of vital proteins, resulting in the orderly demise and removal of the cell.

Animals↗

PubMed1998

Selective regulation of Bcl-XL by a Jak kinase-dependent pathway is bypassed in murine hematopoietic malignancies.

Bcl-2 family proteins are key regulators of apoptosis and function as cell death antagonists (e.g., Bcl-2, Bcl-XL, and Mcl-1) or agonists (e.g., Bax, Bad, and Bak). Here we report that among the Bcl-2 family of proteins tested (Bcl-2, Bcl-XL, Mcl-1, Bax, Bad, and Bak), Bcl-XL was unique in that its protein levels were tightly regulated by hemopoietins in both immortal and primary myeloid progenitors. Investigating signaling pathways utilized by cytokine receptors established that the regulation of Bcl-XL protein levels is mediated by the Jak kinase pathway and is independent of other signaling effectors including STATs, PI-3' kinase, and Ras. Moreover, we provide the first direct evidence that Bcl-X is altered in cancer, because bcl-X expression was activated selectively by retroviral insertions in murine myeloid and T-cell hemopoietic malignancies. Tumors harboring bcl-X insertions had altered bcl-X RNAs, expressed elevated levels of Bcl-XL protein, and lacked the requirements for cytokines normally essential for cell survival. Finally, overexpression of Bcl-XL effectively protected IL-3-dependent myeloid cells from apoptosis following removal of trophic factors. Therefore, Bcl-XL functions as a key cytokine regulated anti-apoptotic protein in myelopoiesis and contributes to leukemia cell survival.

Animals↗

PubMed1998

Regulation of Bcl-xl channel activity by calcium.

Recent studies have demonstrated that the anti-apoptotic proteins, Bcl-2 and Bcl-xl, with the carboxyl-terminal hydrophobic domain removed, form cation-selective channels in the lipid bilayer reconstitution system. However, the regulatory properties of these channels are unknown. In this study, we investigated the ion-conducting properties of full-length Bcl-xl in the lipid bilayer reconstitution system. Our findings indicate that Bcl-xl forms a cation-selective channel that conducts sodium but not calcium and that Bcl-xl channel activity is reversibly inhibited by luminal calcium with a half-dissociation constant of approximately 60 microM. This calcium-dependent regulation of the Bcl-xl channel provides new insights into the roles of calcium and Bcl-2-related proteins in the programmed cell death pathway.

Apoptosis↗

PubMed1998

Linking extracellular survival signals and the apoptotic machinery.

The survival of cells in multicellular organisms requires continuous stimulation from the extracellular environment. The phosphatidylinositol-3' kinase/Akt signaling cascade has been identified as a critical pathway for the transduction of extracellular survival signals. The finding that the pro-apoptotic protein BAD is a substrate of Akt/PKB has provided the first link between extracellular survival signals and the apoptotic machinery.

Animals↗

PubMed1998

EGF receptor signaling inhibits keratinocyte apoptosis: evidence for mediation by Bcl-XL.

Signaling through the epidermal growth factor receptor (EGFR) has been primarily implicated in the growth of epithelial cells including keratinocytes. However, the mechanism by which EGFR stimulation promotes keratinocyte cell growth is poorly understood. Here we report that human keratinocytes undergo apoptosis when incubated with the blocking EGFR monoclonal antibody 225 IgG, or PD153035, a highly specific EGFR tyrosine kinase inhibitor. Endogenous mRNA and protein levels of Bcl-XL, a member the Bcl-2 family which suppresses apoptosis, were specifically inhibited by EGFR blockade. Furthermore, stimulation of EGFR signaling through two natural ligands, transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), increased the expression of Bcl-XL in quiescent keratinocytes and HaCaT cells. Finally, ectopic expression of Bcl-XL in HaCaT cells increased survival after EGFR blockade when compared to untransfected cells or HaCaT keratinocytes transfected with empty vector. These results suggest that the anti-apoptotic protein Bcl-XL plays an important role in the maintenance of keratinocyte survival in response to EGFR signaling.

Antibodies, Monoclonal↗

PubMed1998

E1B 19,000-molecular-weight protein interacts with and inhibits CED-4-dependent, FLICE-mediated apoptosis.

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-xL) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-xL have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.

Adenovirus E1B Proteins↗

PubMed1997

Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors.

Betulinic acid (BA), a melanoma-specific cytotoxic agent, induced apoptosis in neuroectodermal tumors, such as neuroblastoma, medulloblastoma, and Ewing's sarcoma, representing the most common solid tumors of childhood. BA triggered an apoptosis pathway different from the one previously identified for standard chemotherapeutic drugs. BA-induced apoptosis was independent of CD95-ligand/receptor interaction and accumulation of wild-type p53 protein, but it critically depended on activation of caspases (interleukin 1beta-converting enzyme/Ced-3-like proteases). FLICE/MACH (caspase-8), considered to be an upstream protease in the caspase cascade, and the downstream caspase CPP32/YAMA/Apopain (caspase-3) were activated, resulting in cleavage of the prototype substrate of caspases PARP. The broad-spectrum peptide inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cleavage of FLICE and PARP, also completely abrogated BA-triggered apoptosis. Cleavage of caspases was preceded by disturbance of mitochondrial membrane potential and by generation of reactive oxygen species. Overexpression of Bcl-2 and Bcl-XL conferred resistance to BA at the level of mitochondrial dysfunction, protease activation, and nuclear fragmentation. This suggested that mitochondrial alterations were involved in BA-induced activation of caspases. Furthermore, Bax and Bcl-xs, two death-promoting proteins of the Bcl-2 family, were up-regulated following BA treatment. Most importantly, neuroblastoma cells resistant to CD95- and doxorubicin-mediated apoptosis were sensitive to treatment with BA, suggesting that BA may bypass some forms of drug resistance. Because BA exhibited significant antitumor activity on patients' derived neuroblastoma cells ex vivo, BA may be a promising new agent for the treatment of neuroectodermal tumors in vivo.

Antibiotics, Antineoplastic↗

PubMed1997

Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt.

BAD is a distant member of the Bcl-2 family that promotes cell death. Phosphorylation of BAD prevents this. BAD phosphorylation induced by interleukin-3 (IL-3) was inhibited by specific inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Akt, a survival-promoting serine-threonine protein kinase, was activated by IL-3 in a PI 3-kinase-dependent manner. Active, but not inactive, forms of Akt were found to phosphorylate BAD in vivo and in vitro at the same residues that are phosphorylated in response to IL-3. Thus, the proapoptotic function of BAD is regulated by the PI 3-kinase-Akt pathway.

Androstadienes↗

PubMed1997

Bak can accelerate chemotherapy-induced cell death independently of its heterodimerization with Bcl-XL and Bcl-2.

Bak has been shown to both promote apoptosis and to inhibit cell death while two other members of the Bcl-2 family of proteins, Bcl-XL and Bcl-2 delay apoptosis induced by various stimuli including chemotherapeutic agents. We generated clones with stable expression of Bak wild-type (wt) and Bak with its BH3 (delta78-86) domain deleted (deltaBH3) in FL5.12 cells or FL5.12 cells expressing either Bcl-XL or Bcl-2 to determine if Bak could accelerate apoptosis and antagonize the death repressor activity of Bcl-XL and Bcl-2 during chemotherapy-induced apoptosis. We found that Bak accelerated cell death in FL5.12 cells treated with etoposide, fluorouracil or taxol. In FL5.12 cells expressing Bcl-XL and Bak wt or Bak deltaBH3, both Bak wt or Bak deltaBH3 were able to antagonize the protective effect of Bcl-XL when treated with etoposide or fluorouracil. Bak wt or Bak deltaBH3 were also able to abrogate the protective effect of Bcl-2 in cells expressing Bcl-2 and Bak wt or Bak deltaBH3 when challenged by etoposide or fluorouracil. Immunoprecipitation studies revealed that deletion of BH3 disrupted heterodimerization between Bak and Bcl-XL and that both Bak wt and Bak deltaBH3 failed to interact with Bcl-2. These results demonstrate that Bak does not require its BH3 domain to promote apoptosis in stably transfected cells. Furthermore, Bak can accelerate chemotherapy-induced cell death independently of its heterodimerization with Bcl-XL and Bcl-2.

Antineoplastic Agents↗

PubMed1997

Interaction and regulation of the Caenorhabditis elegans death protease CED-3 by CED-4 and CED-9.

In the nematode Caenorhabditis elegans, three genes, ced-3, ced-4, and ced-9, play critical roles in the induction and execution of the death pathway. Genetic studies have suggested that ced-9 controls programmed cell death by regulating ced-4 and ced-3. However, the mechanism by which CED-9 controls the activities of CED-4 and the cysteine protease CED-3, the effector arm of the cell-death pathway, remains poorly understood. Immunoprecipitation analysis demonstrates that CED-9 forms a multimeric protein complex with CED-4 and CED-3 in vivo. Expression of wild-type CED-4 promotes the ability of CED-3 to induce apoptosis in mammalian cells, which is inhibited by CED-9. The pro-apoptotic activity of CED-4 requires the expression of a functional CED-3 protease. Significantly, loss-of-function CED-4 mutants are impaired in their ability to promote CED-3-mediated apoptosis. Expression of CED-4 enhances the proteolytic activation of CED-3. We also show that CED-9 inhibits the formation of p13 and p15, two cleavage products of CED-3 associated with its proteolytic activation in vivo. Moreover, CED-9 inhibits the enzymatic activity of CED-3 promoted by CED-4. Thus, these results provide evidence that CED-4 and CED-9 regulate the activity of CED-3 through physical interactions, which may provide a molecular basis for the control of programmed cell death in C. elegans.

Animals↗

PubMed1997

Bcl-2 and Bcl-XL can differentially block chemotherapy-induced cell death.

Bcl-2 and its homologue Bcl-XL are expressed in a variety of tumors and their expression modulates the sensitivity of tumor cells to a wide spectrum of chemotherapeutic agents and gamma-irradiation. In the present report, we generated clones of FL5.12 lymphoid cells with similar levels of Bcl-2 and Bcl-XL using the Flag epitope to determine if these survival proteins could provide equivalent protection when challenged with chemotherapy or gamma-irradiation. Using four M-phase specific chemotherapeutic agents, Bcl-XL and Bcl-2 provided similar protection against vincristine and vinblastine whereas Bcl-XL afforded as much as 50% greater cell viability than Bcl-2 against etoposide and teniposide-induced cell death. In addition, Bcl-XL provided significantly greater cell viability than Bcl-2 against methotrexate, fluorouracil, and hydroxyurea, three S-phase specific agents. In apoptosis induced by gamma-irradiation and cisplatin, two antitumor treatments that are cell-cycle phase-nonspecific agents, both Bcl-XL and Bcl-2 conferred similar protection against gamma-irradiation, but Bcl-XL provided better protection than Bcl-2 against cisplatin. These results indicate that Bcl-XL and Bcl-2 confer a differential ability to protect against chemotherapy-induced cell death, which appears to be dependent on the molecular mechanism targeted by the drug rather than its cell-cycle phase specificity.

Animals↗

PubMed1997

Bcl-xL overexpression attenuates glutathione depletion in FL5.12 cells following interleukin-3 withdrawal.

Bcl-xL and bax are bcl-2-related genes whose protein products either inhibit or promote apoptosis. Oxidative damage, including the loss of glutathione, has been implicated in the induction of apoptosis. The ability of the Bcl proteins to affect GSH was assessed in control, bax- and bcl-xL-transfected FL5.12 cells [an interleukin (IL)-3-dependent murine prolymphocytic cell line]. Overall levels of GSH were approximately the same in control and bcl-xL transfectants during the 6 h incubation period, although levels increased in bcl-xL transfectants 24 h after replating. GSH in cells overexpressing bax was reduced by approximately 36%. There were no consistent differences between these cell lines in the activities of superoxide dismutase, catalase, glutathione peroxidase or glutathione reductase. Following IL-3 withdrawal, a condition known to cause apoptosis in these cells, a rapid loss of intracellular GSH occurred in control and bax transfectants, which preceded the onset of apoptosis. GSH depletion could not be attributed to intracellular oxidation but rather seemed to occur due to a translocation out of the cell. Cells overexpressing bcl-xL did not lose significant amounts of GSH upon withdrawal of IL-3, and no apoptosis was evident. These results suggest a possible role for GSH in the mechanism by which bcl-xL prevents cell death.

Animals↗

PubMed1997

Genomic organization, promoter region analysis, and chromosome localization of the mouse bcl-x gene.

The bcl-x gene, a bcl-2 family member, is highly regulated during lymphoid development, and its expression modulates apoptosis in lymphoid and other cell populations. Several forms of bcl-x mRNAs with different biologic functions have been described in rodents and humans. In this study, we have determined the organization and promoter region of the mouse bcl-x gene in an effort to understand the molecular basis for the different bcl-x mRNA species identified in tissues. We show that mouse bcl-x maps to the distal mouse chromosome 2 at approximately 89 cM, and exhibits a three-exon structure with an untranslated first exon and a facultative first intron. The coding region of bcl-xL is generated by the juncture of exons II and III through a splicing reaction, whereas bcl-xS is generated by an alternatively utilized donor splice site located within exon II. Analysis of multiple cDNAs and primer extension experiments revealed major transcription initiation sites in brain and thymus within a GC-rich region, with multiple Sp1-binding motifs located upstream of exon I. Another promoter was mapped to a 57-bp region localized upstream of the translation initiation codon by transfection of reporter constructs into FL5.12 and K562 cell lines. The remarkable similarity between the genomic regions of bcl-2 and bcl-x suggests that these genes have evolved from a common ancestral gene or through gene duplication.

Animals↗

PubMed1997

Interaction and regulation of subcellular localization of CED-4 by CED-9.

The Caenorhabditis elegans survival gene ced-9 regulates ced-4 activity and inhibits cell death, but the mechanism by which this occurs is unknown. Through a genetic screen for CED-4-binding proteins, CED-9 was identified as an interacting partner of CED-4. CED-9, but not loss-of-function mutants, associated specifically with CED-4 in yeast or mammalian cells. The CED-9 protein localized primarily to intracellular membranes and the perinuclear region, whereas CED-4 was distributed in the cytosol. Expression of CED-9, but not a mutant lacking the carboxy-terminal hydrophobic domain, targeted CED-4 from the cytosol to intracellular membranes in mammalian cells. Thus, the actions of CED-4 and CED-9 are directly linked, which could provide the basis for the regulation of programmed cell death in C. elegans.

Animals↗

PubMed1997

Quantitation of soluble E-receptor of T lymphocytes in serum from HIV-1 positive patients.

Human T lymphocytes carry a membrane receptor for sheep erythrocytes (E) related to the CD2 molecule. The E-receptor is found in a soluble from (Rs) in serum and can be quantitated by "rocket electrophoresis" using an anti-Rs serum obtained by immunizing sheep with autologous erythrocytes coated with Rs. Increased serum levels of Rs are found in patients with diseases associated with immunodepression. In the present study, 14 asymptomatic HIV-1 seropositive individuals were investigated regarding their Rs levels and delayed hypersensitivity skin tests every 3 months for a period of 35 months. All these patients progressed to AIDS in this period. Rs serum levels have also been quantitated in 14 normal individuals. The mean Rs values in normal individuals, asymptomatic, and AIDS patients were, respectively: 4.8 +/- 1.5 mm (SD), 9.6 +/- 1.9 mm (SD) and 11.3 +/- 2.4 mm (SD). An increase of Rs serum levels was observed when we compared normal individuals with CDC-II and CDC-IV clinical stage patients (P < 0.05, Mann-Whitney test) and CDC-II and CDC-IV patients, (P < 0.05, Wilcoxon test). We have observed a depressed delayed hypersensitivity response to ubiquitous antigens in CDC-IV patients. Our results indicate that Rs serum levels can be used as a progression marker in HIV infected patients.

Adult↗

PubMed1997

Effect of chronic anesthesia on the drug-metabolizing enzyme system and heme pathway regulation.

1. The effect of chronic enflurane or isoflurane anesthesia on hepatic heme regulation and the drug-metabolizing system in mice treated or not with phenobarbital (PB) was investigated. 2. delta-Aminolevulinic acid synthetase was induced 50-170% in all cases. Urinary porphyrin precursor excretion was also enhanced, but these values were lower when animals also received PB. 3. Cytochrome (CYT) P-450 levels were enhanced in animals treated with enflurane whether or not they were given PB. 4. Gluthatione-S-transferase activity was induced by enflurane (138%) or isoflurane (174%), and even more in animals receiving PB also. Sulfatase activity was increased more than 60% with anesthetics. Isoflurane produced a 50% increase of beta-glucuronidase activity and a 35% diminution of tryptophan pyrrolase. 5. The association between anesthetics and PB produced diverse effects on the metabolizing enzyme system. 6. Data suggest that both anesthetics, chemically related, could act through two different mechanisms, however, with the same final effect: heme pathway deregulation.

5-Aminolevulinate Synthetase↗

PubMed1996

Bax homodimerization is not required for Bax to accelerate chemotherapy-induced cell death.

Bax, a member of the Bcl-2 family of proteins, has been shown to accelerate apoptosis induced by growth factor withdrawal, gamma-irradiation, and the chemotherapeutic agent, etoposide. The mechanism by which Bax promotes apoptosis is poorly understood. Bax forms homodimers which have been suggested to act as accelerators or inducers of cell death. However, the requirement for homodimerization of Bax to promote cell death remains unclear. We performed site-directed mutagenesis of the BH1, BH2, and BH3 in Bax to determine the regions of Bax required for homodimerization and to define the role of Bax homodimers in cell death induced by chemotherapy drugs. Bax proteins expressing alanine substitutions of the highly conserved amino acids glycine 108 (G108) in BH1, tryptophan 158 (W158) in BH2, and glycine 67 and aspartic acid 68 (GD67-68) in BH3 as well as deletion of the most conserved amino acids in BH1 (Delta102-112) and BH2 (Delta151-159) and deletion of BH3 (Delta63-71) maintained their ability to accelerate chemotherapy-induced cell death. Immunoprecipitation studies revealed that Bax with deletions in BH1 and BH2 still associated with wild-type Bax while deletion of BH3 disrupted Bax homodimerization. These results demonstrate that Bax does not require the conserved regions of homology, BH1, BH2, or BH3, to accelerate chemotherapy-induced cell death. Furthermore, our results established BH3 as a region required for Bax homodimerization in mammalian cells and demonstrate that monomeric forms of Bax are active in accelerating cell death induced by chemotherapy agents.

Animals↗

PubMed1996

Bax can antagonize Bcl-XL during etoposide and cisplatin-induced cell death independently of its heterodimerization with Bcl-XL.

Bax, a member of the Bcl-2 family of proteins, has been shown to promote apoptosis while other members of the family, including Bcl-XL and Bcl-2, inhibit cell death induced by a variety of stimuli. The mechanism by which Bax promotes cell death is poorly understood. In the present report, we assessed the ability of Bax to antagonize the death repressor activity of Bcl-XL during chemotherapy-induced apoptosis in the lymphoid cell line, FL5.12. Expression of wild-type Bax countered the repressor activity of Bcl-XL against cell death mediated by VP-16 and cisplatin. We performed site-directed mutagenesis of the BH1, BH2, and BH3 homology regions in Bax to determine the ability of wild-type and mutant Bax to heterodimerize with Bcl-XL and to antagonize the protective effect of Bcl-XL against chemotherapy-induced apoptosis. Bax proteins expressing alanine substitutions of the highly conserved amino acids glycine 108 in BH1, tryptophan 151 and 158 in BH2, and glycine 67 and aspartic acid 68 in BH3 retained their ability to promote chemotherapy-induced cell death that was inhibited by Bcl-XL and to form heterodimers with Bcl-XL. Bax proteins containing deletions of the most highly conserved amino acids in BH1 (Delta102-112) and BH2 (Delta151-159) maintained the ability of Bax to antagonize the death repressor activity of Bcl-XL and to associate with Bcl-XL. However, Bax with BH3 deleted did not form heterodimers with Bcl-XL, but retained its ability to counter the death repressor activity of Bcl-XL. These results demonstrate that the conserved BH3, but not BH1 or BH2, homology region of Bax is necessary for its interaction with Bcl-XL in mammalian cells. Furthermore, our results indicate that Bax does not require BH1, BH2, BH3, or heterodimerization with Bcl-XL to counter the death repressor activity of Bcl-XL. Therefore, Bax can antagonize Bcl-XL during VP-16 and, in a lesser degree, during cisplatin-induced cell death independent of its heterodimerization with Bcl-XL.

Amino Acid Sequence↗