Enhancement of UV-induced cytotoxicity by the adeno-associated virus replication proteins.
(17/4256)
Adeno-associated virus (AAV) normally requires co-infection of a helper virus to complete its life cycle. However, under conditions of cellular stress, such as treatment with carcinogens or ultraviolet (UV) light, a permissive intracellular environment is established and AAV completes its replicative cycle producing low levels of progeny virus. AAV DNA replication is dependent upon viral replication proteins, Rep78 and Rep68. The detailed mechanism by which these proteins interact with host cell factors is unknown. We have used a cell line (Neo6) that inducibly expresses the AAV Rep proteins to study their effects on cells that have undergone UV-induced DNA damage. Induction of Rep protein expression immediately after a sub-lethal dose of UV irradiation resulted in rapid cell killing. Those cells that die had chromatin condensation while cellular membranes remained intact, suggesting that concurrent Rep expression and UV damage induces an apoptosis-like response. However, we did not observe any DNA degradation. Thus we believe that the combination of Rep expression and UV irradiation induces cell death that shares some of the characteristics of apoptosis. UV irradiation and Rep expression induced an increase in the level of the CDK inhibitor, p21Cip, and the appearance of modified forms of both p21Cip and Bcl-2. Alteration of normal expression of these cytostatic/apoptotic proteins provides insight into the intracellular targets of the AAV replication proteins. (+info)
Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains.
(18/4256)
This study examined the relationship between blastomere fragmentation in cultured human embryos obtained by in-vitro fertilization and the effect of fragmentation on the distribution of the following eight regulatory proteins found to be: (i) localized in the mature oocyte in subplasmalemmal, polarized domains; and (ii) unequally inherited by the blastomeres during cleavage: leptin, signal transducer and activator of transcription 3 (STAT3), Bax, Bcl-x, transforming growth factor beta 2 (TGF beta 2), vascular endothelial growth factor (VEGF), c-kit and epidermal growth factor R (EGF-R). Four basic patterns of fragmentation were observed. The severity of the impact of each type of fragmentation on the affected blastomere(s) and the developmental competence of the embryo appeared to be a function of the unique temporal and spatial features associated with the particular fragmentation pattern(s) involved in each instance. The findings demonstrate that certain patterns of fragmentation can result in the partial or near total loss of the eight regulatory proteins from specific blastomeres and that the developmental potential of the affected embryo can be particularly compromised if it occurs during the 1- or 2-cell stages. In contrast, fragmentation from portions of a fertilized egg or a blastomere(s) in a 2-cell embryo that do not contain the protein domains, or the complete loss by fragmentation of a regulatory protein domain-containing blastomere after the 4-cell stage does not necessarily preclude continued development to the blastocyst, although the normality and developmental potential of the embryo may be compromised. The possible association between fragmentation and apoptosis was examined by annexin V staining of plasma membrane phosphatidylserine and TUNEL analysis of blastomere DNA. No direct correlation between fragmentation and apoptosis was found following the analyses of fragmented embryos with these two markers. However, while we suggest that changes in cell physiology unrelated to apoptosis are the more likely causes of fragmentation, we cannot exclude the possibility that fragmentation itself may be an initiator of apoptosis if critical ratios or levels of developmentally important proteins are altered by partial or complete elimination of their polarized domains. The findings are discussed with respect to the possible developmental significance of regulatory protein polarization in human oocytes and preimplantation stage embryos. (+info)
Caspase-3 inhibits growth in Saccharomyces cerevisiae without causing cell death.
(19/4256)
Caspase-3, a member of the caspase family of cell death proteases, cleaves cytoplasmic and nuclear substrates and promotes apoptotic cell death in mammalian cells. Although yeast homologs of apoptotic genes have not been identified, some components of apoptotic pathways retain function in yeast. Here we show that the expression of caspase-3 delays cell growth in Saccharomyces cerevesiae without causing cell death. Mutation of the caspase-3 QACRG active site abolished effects on yeast growth. Co-expression of caspase inhibitors alleviated growth inhibition in yeast as did the tripeptide caspase inhibitor ZVAD-fmk. These results suggest that substrates for caspase-3 are present in S. cerevesiae and may participate in the normal cell growth and division processes. (+info)
Investigation of bax-induced release of cytochrome c from yeast mitochondria permeability of mitochondrial membranes, role of VDAC and ATP requirement.
(20/4256)
Recent studies that attempt to explore the action of pro- and anti-apoptotic proteins of the bcl2 family demonstrate the crucial role of relocalization of cytochrome c from the mitochondrial intermembrane space to the cytosol. This early event of apoptosis can be mimicked in the yeast Saccharomyces cerevisiae following expression of bax. In mammalian mitochondria, the mechanism of relocalization is thought to involve the opening of the so-called permeability transition pore. We show in this paper: (a) that bax-induced release of cytochrome c in yeast does not involve any permeability transition of the inner mitochondrial membrane but involves a general alteration of the permeability of the outer mitochondrial membrane to macromolecules. This suggests that a permeability transition of the inner mitochondrial membrane is not an event required for the relocalization of cytochrome c in yeast. (b) The outer-membrane voltage-dependent anion channel (VDAC), a putative component of the permeability transition pore, is not involved in bax-induced release of cytochrome c or in the prevention of this release by bcl-xL. (c) Bax devoid of its C-terminal putative hydrophobic alpha-helix is as efficient as full-length bax to allow the relocalization of cytochrome c, demonstrating this segment of the protein is not required for membrane-targeting. (d) We finally observe that the action of bax on the outer mitochondrial membrane requires the presence of ATP both in vitro and in vivo, and it is shown that ATP directly increases the amount of bax inserted to mitochondria. (+info)
FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis of a human glioblastoma cell line.
(21/4256)
Protein phosphorylation in a human glioblastoma cell line, T98G, was examined after exposure to oxidative stress in vitro. Hydrogen peroxide (1 mM) markedly induced tyrosine phosphorylation of focal adhesion kinase (FAK) and serine phosphorylation of Akt at 1 h after stimulation. Concommitantly, the association of FAK with phosphatidylinositide 3'-OH-kinase (PI 3-kinase) was also observed by the hydrogen peroxide stimulation. When T98G cells were incubated with wortmannin, a PI 3-kinase inhibitor, both PI 3-kinase activity and phosphorylation of Akt were inhibited, whereas apoptosis by oxidative stress was accelerated. Concomitant with apoptosis, elevated level of CPP32 protease activity (caspase-3) was observed, with decreases in Bcl-2 protein and increases in Bax protein. These results suggested that in the signal transduction pathway from FAK to PI 3-kinase, Akt promotes survival. Thus, it became apparent that FAK is the upstream signal protein of the PI 3-kinase-Akt survival pathway in hydrogen peroxide-induced apoptosis in T98G cells. (+info)
Frameshift mutations in TGFbetaRII, IGFIIR, BAX, hMSH3 and hMSH6 are absent in lung cancers.
(22/4256)
A genome-wide instability at simple repeat sequences characterizes gastrointestinal and endometrial cancers of the microsatellite mutator phenotype (MMP). The genes encoding transforming growth factor-beta receptor type II (TGFbetaRII), insulin-like growth factor II receptor (IGFIIR), Bcl-2 associated X protein (BAX), hMSH3 and hMSH6 have simple repeat sequences in their coding regions. Consequently, mutations in the single repeat sequences in these genes provide one major route for carcinogenesis in these cancers. We examined 43 non-small cell lung carcinomas and 16 small cell carcinomas for frameshift mutations in simple repeat sequences of TGFbetaRII, IGFIIR, BAX, hMSH3 and hMSH6. In addition, MMP was assessed using a primer set for BAT-26. None of 59 lung cancers exhibited frameshift mutations or MMP. It is concluded that somatic frameshift mutations in these genes and MMP do not constitute important mechanisms in lung carcinogenesis. The possibility of some sort of genetic instability undetectable as a form of MMP cannot be precluded. (+info)
Apoptosis and expression of Bax, Bcl-x, and Bcl-2 apoptotic regulatory proteins in colorectal carcinomas, and association with p53 genotype/phenotype.
(23/4256)
AIMS: Spontaneous apoptosis and expression of the apoptotic regulatory proteins Bax, Bcl-x, and Bcl-2 were investigated in 50 colorectal carcinomas. The p53 genotypes/phenotypes and BAX genotypes were also determined, and possible associations of these with apoptosis and/or with expression of the different apoptotic regulatory proteins were studied. METHODS: Terminal deoxynucleotidyl transferase (TdT) mediated dUTP labelling of DNA fragments was used to detect apoptotic tumour cells in sections and peroxidase immunohistochemistry was used to assess protein expression. p53 genotype/phenotype was determined using constant denaturant gel electrophoresis/immunoblotting and bax genotype was determined using polymerase chain reaction based methods. RESULTS: The distribution of tumour apoptotic indices was bimodal with a natural cut off at 1.0% (range, 0.0-5.4%); the median fraction of apoptotic tumour cells was 0.8%. Tumour apoptosis was not associated significantly with tumour DNA ploidy status. Normal mucosal tissue had less than 0.1% apoptotic cells. Staining intensities for Bax, Bcl-x, and Bcl-2 were strong; that is, equivalent to or greater than positive normal mucosal cells, in 11 of 50, 20 of 49, and 20 of 48 carcinomas. Frameshift mutations in the bax gene were detected in three of 42 tumours analysed, all of which were DNA diploid, and Bax protein expression in these tumours was absent or very low. Bax, Bcl-x, and Bcl-2 protein expression were not correlated with tumour apoptosis or tumour DNA ploidy status. p53 was expressed in 34 of 50 tumours and p53 gene mutations were detected in 22 of 29 p53 positive tumours analysed. Apoptosis was significantly lower in a greater number of p53 positive tumours than p53 negative tumours. In addition, Bcl-2 protein expression was significantly higher in a greater number of p53 positive tumours compared with p53 negative tumours. Bax and Bcl-x protein expression were not significantly associated with p53 phenotype/genotype. CONCLUSIONS: The results indicate that acquisition of a p53 phenotype is associated with lower spontaneous apoptosis and higher expression of Bcl-2. The results also suggest that p53 is not a major determinant for Bax expression in colorectal carcinomas in vivo. (+info)
Increased BAX expression is associated with an increased risk of relapse in childhood acute lymphocytic leukemia.
(24/4256)
Studies in cell lines have indicated that expression of the BCL-2 family of proteins is an important determinant of chemotherapy-induced apoptosis; however, the level of expression of these proteins in childhood acute lymphoblastic leukemia (ALL) has not been extensively reported. Using quantitative Western blotting we have determined the level of expression of BCL-2, BAX, MCL-1, and BCL-X in lymphoblasts from 47 children with ALL (33 at presentation only, 4 at relapse only, and 10 at both presentation and on relapse). Results were determined as a ratio to actin as an internal control. BCL-2, BAX, and MCL-1 were detected in all samples. BCL-XL was only detected in 6 cases (4 at presentation and 2 at relapse) and BCL-XS in none. No correlation was found between expression and white blood cell count, age at diagnosis, gender, or blast karyotype. BCL-2 levels and the BCL/BAX and MCL-1/BAX ratios were found to be significantly higher in B-lineage as compared with T-lineage disease (P <.003,.02, and.02, respectively). No consistent pattern of change in expression was noted in the 10 cases studied at both presentation and relapse. Kaplan-Meier analysis showed a significant correlation between high BAX expression and an increased probability of relapse (P <.05 by the log rank test), suggesting that chemosensitivity in leukemic blasts may be regulated by factors that override the BCL-2 pathway. (+info)