NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.     

Human VRK2 modulates apoptosis by interaction with Bcl-xL and regulation of BAX gene expression

VRK2 is a novel Ser-Thr kinase whose VRK2A isoform is located in the endoplasmic reticulum and mitochondrial membranes. We have studied the potential role that VRK2A has in the regulation of mitochondrial-mediated apoptosis. VRK2A can regulate the intrinsic apoptotic pathway in two different ways. The VRK2A protein directly interacts with Bcl-xL, but not with Bcl-2, Bax, Bad, PUMA or Binp-3L. VRK2A does not compete with Bax for interaction with Bcl-xL, and these proteins can form a complex that reduces apoptosis. Thus, high VRK2 levels confer protection against apoptosis. In addition, VRK2 knockdown results in an increased expression of BAX gene expression that is mediated by its proximal promoter, thus VRK2A behaves as a negative regulator of BAX. Low levels of VRK2A causes an increase in mitochondrial Bax protein level, leading to an increase in the release of cytochrome C and caspase activation, detected by PARP processing. VRK2A loss results in an increase in cell death that can be detected by an increase in annexinV+ cells. Low levels of VRK2A increase cell sensitivity to induction of apoptosis by chemotherapeutic drugs like camptothecin or doxorubicin. We conclude that VRK2A protein is a novel modulator of apoptosis.     

Halofuginone improves muscle-cell survival in muscular dystrophies

Halofuginone has been shown to prevent fibrosis via the transforming growth factor-β/Smad3 pathway in muscular dystrophies. We hypothesized that halofuginone would reduce apoptosis—the presumed cause of satellite-cell depletion during muscle degradation—in the mdx mouse model of Duchenne muscular dystrophy. Six-week-old mdx mouse diaphragm exhibited fourfold higher numbers of apoptotic nuclei compared with wild-type mice as determined by a TUNEL assay. Apoptotic nuclei were found in macrophages and in Pax7-expressing cells; some were located in centrally-nucleated regenerating myofibers. Halofuginone treatment of mdx mice reduced the apoptotic nuclei number in the diaphragm, together with reduction in Bax and induction in Bcl2 levels in myofibers isolated from these mice. A similar effect was observed when halofuginone was added to cultured myofibers. No apparent effect of halofuginone was observed in wild-type mice. Inhibition of apoptosis or staurosporine-induced apoptosis by halofuginone in mdx primary myoblasts and C2 myogenic cell line, respectively, was reflected by less pyknotic/apoptotic cells and reduced Bax expression. This reduction was reversed by a phosphinositide-3-kinase and mitogen-activated protein kinase/extracellular signal-regulated protein kinase inhibitors, suggesting involvement of these pathways in mediating halofuginone's effects on apoptosis. Halofuginone increased apoptosis in α smooth muscle actin- and prolyl 4-hydroxylase β-expressing cells in mdx diaphragm and in myofibroblasts, the major source of extracellular matrix. The data suggest an additional mechanism by which halofuginone improves muscle pathology and function in muscular dystrophies.     

Expressions of GRP78 and Bax associate with differentiation,metastasis, and apoptosis in non-small cell lung cancer

The purpose of this study was to detect the expressions of GRP78 and Bax in human non-small cell lung cancer (NSCLC) tissues, to analyze their correlations with carcinogenesis and the development of NSCLC, and to investigate the relationship of GRP78 expression to metastasis and apoptosis in the NSCLC cell line HCC827. The positive expression rates of GRP78 and Bax in NSCLC lung tissues were 59.7% and 34.7% by RT-PCR, respectively. The mRNA and protein expression levels of GRP78 in NSCLC tissues were significantly higher than that in the relatively normal surrounding lung tissues (p < 0.05); the lesser the degree of tumor differentiation was, the higher the mRNA and protein expression levels of GRP78 were (p < 0.05). The mRNA and protein expression levels of GRP78 from patients in advanced pathological stages (III–IV) were significantly higher than the corresponding levels in patients in early pathological stages (I–II) (p < 0.05); the mRNA and protein expression levels of GRP78 in patients with positive lymph node metastasis were significantly higher than those in patients with negative lymph node metastasis (p < 0.05). The mRNA and protein expression levels of Bax in the above cases showed the opposite trend of the mRNA and protein expression levels of GRP78. However, the mRNA and protein expression levels of both GRP78 and Bax were independent of the patient’s sex, the patient’s age, the tumor size and the histological type (adenocarcinoma or squamous cell carcinoma) of NSCLC (p > 0.05). The mRNA expression level of GRP78 and the mRNA expression level of Bax in human NSCLC tissues were negatively correlated (r = −0.353, p = 0.002). After transfection of GRP78 siRNA in HCC827 cells, the GRP78 protein expression level was significantly decreased (p < 0.01), while the Bax protein expression level was significantly increased (p < 0.01); the number of cells that passed through the Transwell chamber was significantly less in the non-transfected control group compared to the transfected control group (p < 0.01). The number of apoptotic cells was significantly greater in the non-transfected control group compared to the transfected control group (p < 0.01). The expression levels of GRP78 and Bax were related to the carcinogenesis, development and metastasis of NSCLC. GRP78 expression with siRNA interference in the human NSCLC cell line HCC827 can reduce metastasis and promote apoptosis in HCC827 cells.     

Raf kinase inhibitor protein mediated signaling inhibits invasion and metastasis of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common type of liver cancer with high mortality and poor prognosis. Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways have been implicated in promoting tumor cell proliferation and invasion of HCC cells.MethodsAs a potential inhibitor of tumor metastasis, the role of Raf kinase inhibitor protein (RKIP) in HCC development and the functional relevance with MAPK and NF-κB signaling pathways were investigated. The levels of RKIP expression were examined in human HCC tissues and correlated with tumor stages and metastatic status. Function of RKIP in cellular proliferation, migration, invasion and apoptosis was investigated in HCC cell lines by either overexpressing or knocking down RKIP expression. Mouse xenograft model was established to assess the effect of RKIP expression on tumor growth.ResultsOur results demonstrated decreased RKIP expression in HCC tissues and a strong correlation with tumor grade and distant metastasis. Manipulation of RKIP expression in HCCLM3 and HepG2 cells indicated that RKIP functioned to inhibit HCC cell motility and invasiveness, and contributed to tumor growth inhibition in vivo. Mechanistic studies showed that the function of RKIP was mediated through MAPK and NF-κB signaling pathways. However, cell type-dependent RKIP regulation on these two pathways was also suggested, indicating the complex nature of signaling network.ConclusionOur study provides a better understanding on the molecular mechanisms of HCC metastasis and sets the foundation for the development of targeted therapeutics for HCC.     

Curcumin induces Apaf-1-dependent,p21-mediated caspase activation and apoptosis

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase 3 activation, which is required for apoptosis as confirmed using the pan-caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role for caspase 8 in curcumin-induced caspase 3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase 3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation, both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of pro-apoptotic proteins, such as Bax, Bak, Bid and Bim. Cross-linking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid and Bim causes Bax channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation and apoptosis. Importantly, p21 deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement for p21 in Apaf-1-dependent caspase activation and apoptosis. Together, our findings identify Apaf-1, Bax and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.Key words: curcumin, mitochondria, cytochrome c, Apaf-1, caspase, p21     

Mfn2 Affects Embryo Development via Mitochondrial Dysfunction and Apoptosis

BackgroundGrowth factors, energy sources, and mitochondrial function strongly affect embryo growth and development in vitro. The biological role and prospective significance of the mitofusin gene Mfn2 in the development of preimplantation embryos remain poorly understood. Our goal is to profile the role of Mfn2 in mouse embryos and determine the underlying mechanism of Mfn2 function in embryo development.MethodsWe transfected Mfn2-siRNA into 2-cell fertilized eggs and then examined the expression of Mfn2, the anti-apoptotic protein Bcl-2, and the apoptosis-promoting protein Bax by Western blot. Additionally, we determined the blastocyst formation rate and measured ATP levels, mtDNA levels, mitochondrial membrane potential (ΔΨm), and apoptosis in all of the embryos.ResultsThe results indicate that the Mfn2 and Bcl-2 levels were markedly decreased, whereas Bax levels were increased in the T group (embryos transfected with Mfn2-siRNA) compared with the C group (embryos transfected with control-siRNA). The blastocyst formation rate was significantly decreased in the T group. The ATP content and the relative amounts of mtDNA and cDNA in the T group were significantly reduced compared with the C group. In the T group, ΔΨm and Ca²⁺ levels were reduced, and the number of apoptotic cells was increased.ConclusionLow in vitro expression of Mfn2 attenuates the blastocyst formation rate and cleavage speed in mouse zygotes and causes mitochondrial dysfunction, as confirmed by the ATP and mtDNA levels and mitochondrial membrane potential. Mfn2 deficiency induced apoptosis through the Bcl-2/Bax and Ca²⁺ pathways. These findings indicate that Mfn2 could affect preimplantation embryo development through mitochondrial function and cellular apoptosis.     

Thai Acanthamoeba isolate (T4) induced apoptotic death in neuroblastoma cells via the Bax-mediated pathway

A Thai Acanthamoeba isolate named AS recovered from a corneal scraping of a keratitis patient was genotypically determined as T4. AS trophozoites were used for studying Acanthamoeba-induced apoptosis in mouse neuroblastoma NA cells during in vitro co-cultivation. The Acanthamoeba-exposed NA cells showed signs of apoptosis including cell shrinkage, nuclear condensation and DNA fragmentation. The effect was confirmed by DNA laddering electrophoresis. Involvement of caspase enzymes and mitochondrial pro- and anti-apoptotic proteins (Bax and Bcl-2) in AS-induced apoptosis was determined. The use of Z-VAD-FMK, a pan-caspase inhibitor, significantly reduced the apoptotic effect, while Bax/Bcl-2 ratio analysis showed a significant increase in the expression of apoptotic proteins in AS-exposed NA cells. These results strongly indicated that apoptosis induced by AS trophozoites is caspase-dependent and is mediated by over-expression of pro-apoptotic proteins in the mitochondrial pathway. This is the first report on the role of Bax in mediating apoptosis induced by Acanthamoeba.     

Utilizing mitochondrial events as biomarkers for imaging apoptosis

Cells undergoing apoptosis show a plethora of time-dependent changes. The available tools for imaging apoptosis in live cells rely either on the detection of the activity of caspases, or on the visualization of exposure of phosphatidyl serine in the outer leaflet of the cell membrane. We report here a novel method for the detection of mitochondrial events during apoptosis, namely translocation of Bax to mitochondria and release of cytochrome c (Cyt c) using bimolecular fluorescence complementation. Expression of split yellow fluorescent protein (YFP) fragments fused to Bax and Cyt c, resulted in robust induction of YFP fluorescence at the mitochondria of apoptotic cells with very low background. In vivo expression of split YFP protein fragments in liver hepatocytes and intra-vital imaging of subcutaneous tumor showed elevated YFP fluorescence upon apoptosis induction. Thus, YFP complementation could be applied for high-throughput screening and in vivo molecular imaging of mitochondrial events during apoptosis.     

Bax Activates Endophilin B1 Oligomerization and Lipid Membrane Vesiculation

Endophilins participate in membrane scission events that occur during endocytosis and intracellular organelle biogenesis through the combined activity of an N-terminal BAR domain that interacts with membranes and a C-terminal SH3 domain that mediates protein binding. Endophilin B1 (Endo B1) was identified to bind Bax, a Bcl-2 family member that promotes apoptosis, through yeast two-hybrid protein screens. Although Endo B1 does not bind Bax in healthy cells, during apoptosis, Endo B1 interacts transiently with Bax and promotes cytochrome c release from mitochondria. To explore the molecular mechanism of action of Endo B1, we have analyzed its interaction with Bax in cell-free systems. Purified recombinant Endo B1 in solution displays a Stokes radius indicating a tetrameric quarternary structure. However, when incubated with purified Bax, it assembles into oligomers more than 4-fold greater in molecular weight. Although Endo B1 oligomerization is induced by Bax, Bax does not stably associate with the high molecular weight Endo B1 complex. Endo B1 oligomerization requires its C-terminal Src homology 3 domain and is not induced by Bcl-xL. Endo B1 combined with Bax reduces the size and changes the morphology of giant unilamellar vesicles by inducing massive vesiculation of liposomes. This activity of purified Bax protein to induce cell-free assembly of Endo B1 may reflect its activity in cells that regulates apoptosis and/or mitochondrial fusion.     

mED2—A Novel Gene Involved in Mouse Embryonic Development

Dissection of new genes underlying embryonic development is important for our understanding of the molecular mechanism of vertebrate embryonic development. In this study, the expression pattern and functional analysis of a new gene, called mED2, originally cloned from mouse embryos using subtractive hybridization was reported. mED2 expression patterns were characterized by RT-PCR-Southern hybridization and in situ hybridization. The results showed that mED2 was mainly expressed in the embryonic nervous system and mesoderm-derived tissues and its expression varied depending on the embryonic developmental stages. The knockdown of mED2 activity by antisense RNA injection inhibited zygote cleavage and blastocyst formation during pre-implantation in mice. Subcellular localization of mED2-eGFP fusion protein revealed a pattern of nuclear membrane and juxta-/perinuclear location such as in the rough endoplasmic reticulum and Golgi apparatus. This finding was supported by bioinformatics analysis, which indicated mED2 protein to be a transmembrane protein with partial homology to the thioredoxin family of proteins. It is inferred that mED2 gene can probably take part in early embryonic development in mouse and may be involved in target protein posttranslational modification, turnover, folding, and stability at the endoplasmic reticulum and/or the Golgi apparatus.