Bcl-X(L) translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

BACKGROUND: The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X(L), pro-apoptotic Bax and Bad). METHODS: Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (Western immunoblots, densitometry, immunoelectron microscopy). RESULTS: Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X(L) and Bax, but not Bcl-2 or Bad, was identified in control distal cells. Bcl-X(L) and Bax had nonsignificant increases (P> 0.05) in these cells. Bcl-2, Bax, and Bcl-X(L), but not Bad, were endogenously expressed in control proximal cells. Bcl-X(L) was significantly decreased in treated proximal cultures (P < 0.05), with Bax and Bcl-2 having nonsignificant increases (P> 0.05). Immunoelectron microscopy localization indicated that control and treated but surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X(L) from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-X(L) expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. CONCLUSION: The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X(L) in proximal cells, as well as translocation of Bcl-X(L) protein to mitochondria within the surviving distal cells.

Escape from apoptosis after prolonged serum deprivation is associated with the regulation of the mitochondrial death pathway by Bcl-x(l)

Bcl-x(l) and Bax play important roles in the regulation of apoptosis. This study investigated the involvement of the mitochondrial death pathway and the role of Bcl-x(l) and Bax in the escape from apoptosis after prolonged serum deprivation in Madin-Darby canine kidney (MDCK) cells. Low level apoptosis and basal activity of the mitochondrial death pathway were detectable in normal cell growth. In serum deprivation, mitosis was partially suppressed, and the mitochondrial activity was stimulated. The level of apoptosis continuously rose over 48 h. This rise was concomitant with the increasing presence of cytochrome c in cytosol. However, both apoptosis and cytosolic cytochrome c fell dramatically at 72 h. Elevation of whole cell Bcl-x(l) and redistribution of Bcl-x(l) protein from cytosol to the membrane at 48 h and 72 h was observed. Redistribution of Bax protein from the membrane to cytosol occurred at 24 h, and remained steady to 72 h. Bax/Bcl-x(l) coimmunoprecipitation by anti-Bax antibody showed reduced Bax/Bcl-x(l) interaction at the membrane at 72 h, but not at 24 or 48 h. These results suggest that apoptosis upon serum withdrawal results from the leakage of cytochrome c to cytosol. Amelioration of the leakage of cytochrome c and apoptosis requires not only the increase of Bcl-x(l)/Bax ratio, but also the release of Bcl-x(l) from Bax at the membrane.

Modulation of BCL-X$\sb{\rm L}$ is a critical determinant of c-myc induced apoptosis

The fine balance between proliferation and apoptosis plays a primary role in carcinogenesis. Proto-oncogenes that induce both proliferation and apoptosis provide a powerful inbuilt system to inhibit clonal expansion of cells with high proliferation rates. This provides a restraint to the development of neoplasms. C-myc expressing cells undergo apoptosis in low serum by an unknown mechanism. Several lines of evidence suggested that c-myc induces apoptosis by a transcriptional mechanism. However, the target genes of this program have not been fully defined. Protein synthesis inhibitors induce apoptosis in c-myc over-expressing cells at high serum levels suggesting that inhibition of synthesis of a survival factor may induce apoptosis. We show that the expression of c-myc directly correlates with an increase in the level of a survival protein, bcl-$\rm x\sb{L},$ and a decrease in the pro-apoptotic protein, bax, at both the protein and mRNA level. Furthermore, a significant decrease of the bcl-$\rm x\sb{L}$ protein levels is observed under low serum conditions. In order to investigate the mechanism of regulation of bcl-$\rm x\sb{L}$ and bax by c-myc, the bcl-x and bax promoters were cloned, sequenced and shown to contain c-myc binding sites. The chloramephenicol acetyl transferase (CAT) reporter assay was used to demonstrate activation of the bcl-x promoter by increasing levels of c-myc when co-transfected in COS cells. The bax promoter was also shown to be transrepressed in c-myc expressing cells. The role of bcl-$\rm x\sb{L}$ in apoptosis regulation in c-myc cell lines in normal and low serum was then investigated. Cells lines expressing c-myc and bcl-$\rm x\sb{L}$ were generated and were shown to be resistant to apoptosis induction in low serum. Furthermore, cell lines expressing c-myc, anti-sense bcl-$\rm x\sb{L}$ and $\beta$-galactosidase demonstrated significantly enhanced rates of apoptosis in high serum compared to c-myc Rat 1a cells. These findings suggest that c-myc activates a survival program involving bcl-$\rm x\sb{L}$ upregulation and bax downregulation. However, this survival signal is reduced under low serum conditions by the relative downregulation of bcl-$\rm x\sb{L}$ allowing for apoptosis to proceed. These data also directly demonstrates that downregulation in the level of bcl-$\rm x\sb{L}$ associated with low serum conditions is a critical determinant of c-myc induced apoptosis. ^

The reduced expression of the HADH2 protein causes X-linked mental retardation, choreoathetosis, and abnormal behavior

Recently, we defined a new syndromic form of X-linked mental retardation in a 4-generation family with a unique clinical phenotype characterized by mild mental retardation, choreoathetosis, and abnormal behavior (MRXS10). Linkage analysis in this family revealed a candidate region of 13.4 Mb between markers DXS1201 and DXS991 on Xp11; therefore, mutation analysis was performed by direct sequencing in most of the 135 annotated genes located in the region. The gene (HADH2) encoding L-3-hydroxyacyl-CoA dehydrogenase II displayed a sequence alteration (c.574 C-->A; p.R192R) in all patients and carrier females that was absent in unaffected male family members and could not be found in 2,500 control X chromosomes, including in those of 500 healthy males. The silent C-->A substitution is located in exon 5 and was shown by western blot to reduce the amount of HADH2 protein by 60%-70% in the patient. Quantitative in vivo and in vitro expression studies revealed a ratio of splicing transcript amounts different from those normally seen in controls. Apparently, the reduced expression of the wild-type fragment, which results in the decreased protein expression, rather than the increased amount of aberrant splicing fragments of the HADH2 gene, is pathogenic. Our data therefore strongly suggest that reduced expression of the HADH2 protein causes MRXS10, a phenotype different from that caused by 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, which is a neurodegenerative disorder caused by missense mutations in this multifunctional protein.

Selective induction of apoptosis by the pyrrolo-1,5-benzoxazepine 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) in Leukemia cells occurs via the c-Jun NH2-terminal kinase-dependent phosphorylation and inactivation of Bcl-2 and Bcl-XL

Overexpression of the Bcl-2 proto-oncogene in tumor cells confers resistance against chemotherapeutic drugs. In this study, we describe how the novel pyrrolo-1,5-benzoxazepine compound 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) selectively induces apoptosis in Bcl-2-overexpressing cancer cells, whereas it shows no cytotoxic effect on normal peripheral blood mononuclear cells. PBOX-6 overcomes Bcl-2-mediated resistance to apoptosis in chronic myelogenous leukemia (CML) K562 cells by the time- and dose-dependent phosphorylation and inactivation of antiapoptotic Bcl-2 family members Bcl-2 and Bcl-XL. PBOX-6 also induces Bcl-2 phosphorylation and apoptosis in wild-type T leukemia CEM cells and cells overexpressing Bcl-2. This is in contrast to chemotherapeutic agents such as etoposide, actinomycin D, and ultraviolet irradiation, whereby overexpression of Bcl-2 confers resistance against apoptosis. In addition, PBOX-6 induces Bcl-2 phosphorylation and apoptosis in wild-type Jurkat acute lymphoblastic leukemia cells and cells overexpressing Bcl-2. However, Jurkat cells containing a Bcl-2 triple mutant, whereby the principal Bcl-2 phosphorylation sites are mutated to alanine, demonstrate resistance against Bcl-2 phosphorylation and apoptosis. PBOX-6 also induces the early and transient activation of c-Jun NH2-terminal kinase (JNK) in CEM cells. Inhibition of JNK activity prevents Bcl-2 phosphorylation and apoptosis, implicating JNK in the upstream signaling pathway leading to Bcl-2 phosphorylation. Collectively, these findings identify Bcl-2 phosphorylation and inactivation as a critical step in the apoptotic pathway induced by PBOX-6 and highlight its potential as an effective antileukemic agent.

Channel formation by antiapoptotic protein Bcl-2

Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic α-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (Δh5, 6) mutant did not. The most frequent conductance observed (18 ± 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 ± 2 pS and 90 ± 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.

Solution structure of the antiapoptotic protein bcl-2

The structures of two isoforms of Bcl-2 that differ by two amino acids have been determined by NMR spectroscopy. Because wild-type Bcl-2 behaved poorly in solution, the structures were determined by using Bcl-2/Bcl-xL chimeras in which part of the putative unstructured loop of Bcl-2 was replaced with a shortened loop from Bcl-xL. These chimeric proteins have a low pI compared with the wild-type protein and are soluble. The structures of the two Bcl-2 isoforms consist of 6 α-helices with a hydrophobic groove on the surface similar to that observed for the homologous protein, Bcl-xL. Comparison of the Bcl-2 structures to that of Bcl-xL shows that although the overall fold is the same, there are differences in the structural topology and electrostatic potential of the binding groove. Although the structures of the two isoforms of Bcl-2 are virtually identical, differences were observed in the ability of the proteins to bind to a 25-residue peptide from the proapoptotic Bad protein and a 16-residue peptide from the proapoptotic Bak protein. These results suggest that there are subtle differences in the hydrophobic binding groove in Bcl-2 that may translate into differences in antiapoptotic activity for the two isoforms.

A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

bcl-x is expressed in embryonic and postnatal neural tissues and functions to prevent neuronal cell death.

Previous studies have implicated the bcl-2 protooncogene as a potential regulator of neuronal survival. However, mice lacking functional bcl-2 exhibited normal development and maintenance of the central nervous system (CNS). Since bcl-2 appears dispensable for neuronal survival, we have examined the expression and function of bcl-x, another member of the bcl-2 family of death regulatory genes. Bcl-2 is expressed in neuronal tissues during embryonic development but is down-regulated in the adult CNS. In contrast, Bcl-xL expression is retained in neurons of the adult CNS. Two different forms of bcl-x mRNA and their corresponding products, Bcl-xL and Bcl-x beta, were expressed in embryonic and adult neurons of the CNS. Microinjection of bcl-xL and bcl-x beta cDNAs into primary sympathetic neurons inhibited their death induced by nerve growth factor withdrawal. Thus, Bcl-x proteins appear to play an important role in the regulation of neuronal survival in the adult CNS.

Apoptose em otorrinolaringologia e cabeça e pescoço

This paper intend to review recent advances in our understanding of programmed cell death, or apoptosis, and discuss implications of these basic science advances in the development of causes and potential treatments of a variety of diseases of the head and neck. Conclusions: apoptosis is now understood to be important in the normal development and survival of all multicellular organism. Deregulation of this normally tighly controlled process underlies a variety of disease states, including neoplasia, autoimmune disease, and disorders of the central nervous system. A better understanding of this process and regulation may help otolaryngologists better understand diseases relevant to this specialty and will lead to improved therapeutic interventions.

Regulation of Bcl-xL expression by H2O2 in cardiac myocytes.

Oxidative stress promotes cardiac myocyte apoptosis through the mitochondrial death pathway. Since Bcl-2 family proteins are key regulators of apoptosis, we examined the effects of H2O2 on the expression of principal Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bad) in neonatal rat cardiac myocytes. Protein expression was assessed by immunoblotting. Bcl-2, Bax, and Bad were all down-regulated in myocytes exposed to 0.2 mm H2O2, a concentration that induces apoptosis. In contrast, although Bcl-xL levels initially declined, the protein was re-expressed from 4-6 h. Bcl-xL mRNA was up-regulated from 2 to 4 h in neonatal rat or mouse cardiac myocytes exposed to H2O2, consistent with the re-expression of protein. Four different untranslated first exons have been identified for the Bcl-x gene (exons 1, 1B, 1C, and 1D, where exon 1 is the most proximal and exon 1D the most distal to the coding region). All were detected in mouse or rat neonatal cardiac myocytes, but exon 1D was not expressed in adult mouse hearts. In neonatal mouse or rat cardiac myocytes, H2O2 induced the expression of exons 1B, 1C, and 1D, but not exon 1. These data demonstrate that the Bcl-x gene is selectively responsive to oxidative stress, and the response is mediated through distal promoter regions.

Analysis of Ki-67 and Bcl-2 protein expression in normal colorectal mucosa of women with breast cancer

The aim of this study was to evaluate Ki-67 and Bcl-2 protein expression in the normal colorectal mucosa adjacent to adenomatous polyps in women with breast cancer. A cross-sectional, controlled study was conducted in 35 women with and without breast cancer who had adenomatous colorectal polyps. The patients were divided into two groups: Group A (a control group of women without breast cancer, n = 18) and Group B (a study group of women with breast cancer, n = 17). A sample of normal colonic mucosa was collected at a distance of 5 cm from the polypoid lesion to evaluate immunchistochemical expression of the Ki-67 and Bcl-2 proteins. Student`s t-test and the chi-square test were used to analyse Ki-67 and Bcl-2 expression, respectively. Statistical significance was established at p < 0.05. The mean percentage of Ki-67-stained nuclei in Groups A and B was 25.12 +/- 2.08 and 41.50 +/- 1.85, respectively (p < 0.001), whereas the percentage of cases with cells expressing Bcl-2 in Groups A and B was 17.6% and 82.4%, respectively (p < 0.003). In the present study, greater proliferative activity and greater expression of the antiapoptotic protein Bcl-2 was found in the normal colorectal mucosa of women with breast cancer. (C) 2009 Elsevier Ltd. All rights reserved.

Immunohistochemical expression of PCNA, p53, bax and bcl-2 in oral lichen planus and epithelial dysplasia.

The potential for malignant transformation of oral lichen planus is still controversial. The expression of proteins related to cell proliferation and apoptosis in oral lichen planus and epithelial dysplasia was analyzed to evaluate the true potential for malignant transformation of this disease. Twenty-four cases of each lesion were subjected to the streptoavidin-biotin technique for identifying the immunohistochemical expression of PCNA, p53, bax, and bcl-2 proteins. Of the 24 cases of oral lichen planus, 14 (58.33%) were positive for PCNA, 10 (41.67%) for p53, 4 (16.67%) for bcl-2 and 12 (50%) for bax, whereas of the 24 cases of epithelial dysplasia, 20 (83.33%) were positive for PCNA, 10 (41.67%) for p53, 6 (25%) for bcl-2, and 20 (83.33%) for bax. Chi-squared test showed no statistically significant differences between the expression of p53 and bcl-2 in oral lichen planus and epithelial dysplasia, regardless of the grade (P > 0.05). However, the expression of PCNA and bax was significantly increased in epithelial dysplasia (P < 0.05). The results of this study showed that alterations in expression of these proteins are observed in oral lichen planus and epithelial dysplasia, suggesting the potential for malignant transformation in both lesions.

Correlation between immunoexpression of BCL-2 protein family with apoptosis index, cellular proliferation and survival in colorectal carcinomas

Objectives: To evaluate the Bcl-2, Bax, Bad and Bak immunoexpression in tumor and nontumorous tissue of 130 patients with colorectal carcinoma submitted to surgery at São Paulo Hospital, EPM/ UNIFESP, from 2002 to 2005, and to correlate the immunoexpression data with the apoptotic index (AI, obtained by anti-cleaved caspase 3 and M30 labeling), cell proliferation score (CPS, obtained by Ki-67), immunoexpression of p53 and patient’s clinical prognosis. Results: Positive correlation was verified between Bcl-2 protein family in tumor and nontumor tissue. Only Bcl-2 protein correlated with IA and CPS in the tumor. Positive correlation was observed between pro- -apoptotic proteins and Bcl-2 protein. In the adjacent mucosa, Bcl-2 correlated with Ki-67 and p53, but not with IA. Carcinomas exhibited higher immunoexpression of CPS and IA markers. No correlation occurred between immunoexpression data and patient survival. Conclusion: Positive correlation was observed between the pro-apoptotic proteins of the Bcl-2 family and the anti-apoptotic protein Bcl-2. In the adjacent nontumor mucosa, Bcl-2 correlated with Ki-67 and p53, but not with AI. Carcinomas presented greater immunoexpression for CPS and AI markers; however immunoexpression of these markers was not correlated with patient survival.