Mcl-1 is a potential therapeutic target in multiple types of cancer

Resistance to apoptosis is a common challenge in human malignancies contributing to both progress of cancer and resistance to conventional therapeutics. Abnormalities in a variety of cell intrinsic and extrinsic molecular mechanisms cooperatively promote tumor formation. Therapeutic approaches that specifically target components of these molecular mechanisms are getting widespread attention. Mcl-1 is a highly expressed pro-survival protein in human malignancies and its cellular expression is tightly regulated via multiple mechanisms. Mcl-1 differs from other members of the Bcl-2 family in having a very short half-life. So inhibition of its expression and/or neutralization of its anti-apoptotic function will rapidly make Mcl-1-dependent cells more susceptible to apoptosis and provide an opportunity to combat several types of cancers. This review summarizes the current knowledge on the regulation of Mcl-1 expression and discusses the alternative approaches targeting Mcl-1 in human cancer cells whose survivals mainly depend on Mcl-1. Received 6 October 2008; received after revision 21 October 2008; accepted 10 November 2008     

Fank1 interacts with Jab1 and regulates cell apoptosis via the AP-1 pathway

Regulation of apoptosis at various stages of differentiation plays an important role in spermatogenesis. Therefore, the identification and characterisation of highly expressed genes in the testis that are involved in apoptosis is of great value to delineate the mechanism of spermatogenesis. Here, we reported that Fank1, a novel gene highly expressed in testis, functioned as an anti-apoptotic protein that activated the activator protein 1 (AP-1) pathway. We found that Jab1 (Jun activation domain-binding protein 1), a co-activator of AP-1, specifically interacted with Fank1. Reporter analyses showed that Fank1 activated AP-1 pathway in a Jab1-dependent manner. Fank1 overexpression also increased the expression and activation of endogenous c-Jun. Further study showed that Fank1 inhibited cell apoptosis by upregulating and activating endogenous c-Jun and its downstream target, Bcl-3. This process was shown to be Jab1 dependent. Taken together, our results indicated that by interacting with Jab1, Fank1 could suppress cell apoptosis by activating the AP-1-induced anti-apoptotic pathway.     

Cyclin D3 and p53 mediate sulforaphane-induced cell cycle delay and apoptosis in non-transformed human T lymphocytes

Despite experimental evidence that sulforaphane can exert chemopreventive effects, whether these effects are specific for neoplastic cells is not known. Following our previous demonstration that sulforaphane induces cell cycle arrest and apoptosis in human T lymphoblastoid Jurkat leukemia cells and increases p53 and bax protein expression, we tested sulforaphane on non-transformed phytohemagglutinin-stimulated human lymphocytes. Here, we demonstrate that sulforaphane arrested cell cycle progression in G, phase, through a decrease in the protein expression of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53. These findings suggest that sulforaphane is a growth modulator for T cells. Our in vitro evidence that sulforaphane is active and even cytotoxic in normal as well as transformed lymphocytes raises important questions regarding its suitability for cancer chemoprevention.     

Cyclin A1 is a p53-induced gene that mediates apoptosis, G2/ M arrest, and mitotic catastrophe in renal, ovarian, and lung carcinoma cells

We were the first to identify cyclin A1 as a p53-induced gene by cDNA expression profiling of p53-sensitive and -resistant tumor cells [Maxwell S. A. and Davis G. E. (2000) Proc. Natl. Acad. Sci. USA 97, 13009–13014]. We show here that cyclin A1 can induce G2 cell cycle arrest, polyploidy, apoptosis, and mitotic catastrophe in H1299 non-small cell lung, TOV-21G ovarian, or 786-0 renal carcinoma cells. More cdk1 protein and kinase activities were observed in cyclin A1-induced cells than in GFP control-induced cells. Thus, cyclin A1 might mediate apoptosis and mitotic catastrophe through an unscheduled or inappropriate activation of cdk1. Two primary renal cell carcinomas expressing mutated p53 exhibited reduced or absent expression of cyclin A1 relative to the corresponding normal tissue. Moreover, renal carcinoma-derived mutant p53s were deficient in inducing cyclin A1 expression in p53-null cells. Cyclin A1 but not cyclin A2 was upregulated in etoposide-treated tumor cells undergoing p53-dependent apoptosis and mitotic catastrophe. Forced upregulation of cyclin A2 did not induce apoptosis. The data implicate cyclin A1 as a downstream player in p53-dependent apoptosis and G2 arrest. Received 1 November 2005; received after revision 17 February 2006; accepted 13 April 2006     

Interaction of DISC1 with the PTB domain of Tensin2

The gene for Disrupted-in-Schizophrenia 1 (DISC1) is amongst the most significant risk genes for schizophrenia. The DISC1 protein is an intracellular scaffolding molecule thought to act an important hub for protein interactions involved in signalling for neural cell differentiation and function. Tensin2 is an intracellular actin-binding protein that bridges the intracellular portion of transmembrane receptors to the cytoskeleton, thereby regulating signalling for cell shape and motility. In this study, we probed in molecular detail a novel interaction between DISC1 and Tensin2. Western blot and confocal microscopic analyses revealed widespread expression of both DISC1 and Tensin2 proteins throughout the mouse brain. Furthermore, we have developed novel anti-DISC1 antibodies that verified the predominant expression of a 105-kDa isoform of DISC1 in the rodent brain as well as in human cells. In the mouse brain, both proteins showed region-specific expression patterns, including strong expression in the pyramidal cell layer of the hippocampus and dentate gyrus. DISC1–Tensin2 colocalisation was most clearly observed in the Purkinje cells of the mouse cerebellum. Biochemical coimmunoprecipitation experiments revealed an interaction between endogenous DISC1 and Tensin2 proteins in the mouse brain. Further pulldown studies in human cells using Myc-tagged Tensin2 constructs revealed that DISC1 specifically interacts with the C-terminal PTB domain of Tensin2 in a phosphorylation-independent manner. This new knowledge on the DISC1–Tensin2 interaction, as part of the wider DISC1 interactome, should further elucidate the signalling pathways that are perturbed in schizophrenia and other mental disorders.     

Experimental verification of a conserved intronic microRNA located in the human <Emphasis Type="Italic">TrkC</Emphasis> gene with a cell type-dependent apoptotic function

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis induction and tumorigenesis. We hypothesized that, similar to p75^NTR receptor, some of the diverse functions of TrkC could be mediated by a microRNA (miRNA) embedded within the gene. Here, we experimentally verified the expression and processing of two bioinformatically predicted miRNAs named TrkC-miR1-5p and TrkC-miR1-3p. Transfecting a DNA fragment corresponding to the TrkC-premir1 sequence in HEK293t cells caused ~300-fold elevation in the level of mature TrkC-miR1 and also a significant downregulation of its predicted target genes. Furthermore, endogenous TrkC-miR1 was detected in several cell lines and brain tumors confirming its endogenous generation. Furthermore, its orthologous miRNA was detected in developing rat brain. Accordingly, TrkC-miR1 expression was increased during the course of neural differentiation of NT2 cell, whereas its suppression attenuated NT2 differentiation. Consistent with opposite functions of TrkC, TrkC-miR1 overexpression promoted survival and apoptosis in U87 and HEK293t cell lines, respectively. In conclusion, our data report the discovery of a new miRNA with overlapping function to TrkC.     

Unscheduled CDK1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells

Cyclin-dependent kinase 1 (CDK1) is a major component of the cell cycle progression engine. Recently, several investigations provided evidence demonstrating that unscheduled CDK1 activation may also be involved in apoptosis in cancerous cells. In this article, we demonstrate that X-ray irradiation induced G1 arrest in MOLT-4 lymphocytic leukemia cells, the arrest being accompanied by reduction in the activity of CDK2, but increased CDK1 activity and cell apoptosis in the G1 phase. Interestingly, this increase in CDK1 and apoptosis by ionizing radiation was prevented by pretreatment with the CDK1 inhibitor, roscovitine, suggesting that CDK1 kinase activity is required for radiation-induced apoptotic cell death in this model system. Furthermore, cyclin B1 and CDK1 were detected co-localizing and associating in G1 phase MOLT-4 cells, with the cellular lysates from these cells revealing a genotoxic stress-induced increase in CDK1 phosphorylation (Thr-161) and dephosphorylation (Tyr-15), as analyzed by postsorting immunoprecipitation and immunoblotting. Finally, X-irradiation was found to increase Bcl-2 phosphorylation in G1 phase cells. Taken together, these novel findings suggest that CDK1 is activated by unscheduled accumulation of cyclin B1 in G1 phase cells exposed to X-ray, and that CDK1 activation, at the wrong time and in the wrong phase, may directly or indirectly trigger a Bcl-2-dependent signaling pathway leading to apoptotic cell death in MOLT-4 cells. Received 30 March 2006; received after revision 23 June 2006; accepted 24 August 2006 J. Wu and Y. Feng contributed equally to this work.     

A Smurf1 tale: function and regulation of an ubiquitin ligase in multiple cellular networks

Since being discovered and intensively studied for over a decade, Smad ubiquitylation regulatory factor-1 (Smurf1) has been linked with several important biological pathways, including the bone morphogenetic protein pathway, the non-canonical Wnt pathway, and the mitogen-activated protein kinase pathway. Multiple functions of this ubiquitin ligase have been discovered in cell growth and morphogenesis, cell migration, cell polarity, and autophagy. Smurf1 is related to physiological manifestations in terms of age-dependent deficiency in bone formation and invasion of tumor cells. Smurf1-knockout mice have a significant phenotype in the skeletal system and considerable manifestations during embryonic development and neural outgrowth. In depth studying of Smurf1 will help us to understand the etiopathological mechanisms of related disorders. Here, we will summarize historical and recent studies on Smurf1, and discuss the E3 ligase-dependent and -independent functions of Smurf1. Moreover, intracellular regulations of Smurf1 and related physiological phenotypes will be described in this review.     

Functional mechanisms of the cellular prion protein (PrPC) associated anti-HIV-1 properties

The cellular prion protein PrP(C)/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrP(C) displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrP(C) displays important similarities with the HIV-1 nucleocapsid protein and found that PrP(C) expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrP(C) mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrP(C) binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrP(C) colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrP(C) in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.     

Proteases in apoptosis

The interleukin-1 β-converting enzyme (ICE)-like family proteases have recently been identified as key enzymes in apoptotic cell death. Among these proteases one can identify specific activities which may be involved in cytokine production or in resident protein cleavage. Several factors influence the constitutive apoptotic mechanism and may provide insight into the role of protease(s) in apoptosis. Although it appears that ICE family members play a most important role in promoting apoptotic cell death, evidence has been advanced that other proteases are also involved in sequential or parallel steps of apoptosis. Activation of a particular protease can lead to processing molecules either of the same or different proteases, leading to an activation of a protease cascade. Here we attempt to summarize the current thinking concerning these proteases and their involvement in apoptosis.     

Survivin过表达对MS1细胞Survivin基因及蛋白表达与凋亡的作用

目的 探讨Survivin过量表达对MS1细胞Survivin基因及蛋白表达与凋亡的影响.方法 构建Survivin真核表达载体,以脂质体转染MS1细胞,采用流式细胞仪的方法检测细胞凋亡;Real-time PCR和Western blot检测Survivin的mRNA及蛋白水平.结果 转染Survivin实验组与空载体对照组比较,Survivin mRNA和蛋白表达明显增高,实验组细胞凋亡率较对照组下降.结论 Survivin过表达可使MS1细胞的Survivin蛋白及mRNA高表达并能使细胞凋亡凋亡率下降,为后续的动物实验打下了理论依据.     

Proteasome inhibition overcomes the resistance of renal cell carcinoma cells against the PPARγ ligand troglitazone

In order to analyze the effects of peroxisome proliferator-activated receptor-γ (PPARγ) activation on renal cell carcinomas we utilized several cell lines that were treated with the high affinity PPARγ agonist, troglitazone. Incubation of RCC cells with troglitazone resulted in reduced secretion of growth factors that was due to the inhibition of MAP kinase signaling and reduced nuclear localized expression of relB and HIF1alpha. Interestingly, the cell lines used showed a different sensitivity towards apoptosis induction that did not correlate with the inhibition of growth factors or expression of pro- and antiapoptotic molecules. To overcome this resistance the cells were treated with a combination of troglitazone and the proteasome inhibitor, bortezomib. The combination of both compounds induced apoptosis even in cells resistant to both agents alone, due to increased induction of ER-stress and caspase-3 mediated cell death. Received 03 September 2009; received after revision 02 February 2009; accepted 10 February 2009     

Reduced syncytin-1 expression in choriocarcinoma BeWo cells activates the calpain1–AIF-mediated apoptosis,implication for preeclampsia

Placentas associated with preeclampsia are characterized by extensive apoptosis in trophoblast lineages. Syncytin-1 (HERVWE1) mediates the fusion of cytotrophoblasts to form syncytiotrophoblasts, which assume the placental barrier, fetal–maternal exchange and endocrine functions. While decreased syncytin-1 expression has been observed in preeclamptic placentas, it is not clear if this alteration is involved in trophoblast apoptosis. In the current study, we found that siRNA-mediated knockdown of syncytin-1 led to apoptosis in choriocarcinoma BeWo, a cell line of trophoblastic origin. Characterization of the apoptotic pathways indicated that this effect does not rely on the activation of caspases. Rather, decreased syncytin-1 levels activated the apoptosis inducing factor (AIF) apoptotic pathway by inducing the expression, cleavage, and nuclear translocation of AIF. Moreover, calpain1, the cysteine protease capable of cleaving AIF, was upregulated by syncytin-1 knockdown. Furthermore, treatment with calpain1 inhibitor MDL28170 effectively reversed AIF cleavage, AIF nuclear translocation, and cell apoptosis triggered by syncytin-1 downregulation, verifying the specific action of calpain1–AIF pathway in trophoblast apoptosis. We confirmed that preeclamptic placentas express lower levels of syncytin-1 than normal placentas, and observed an inverse correlation between syncytin-1 and AIF/calpain1 mRNA levels, a result consistent with the in vitro findings. Immunohistochemistry analyses indicated decreased syncytin-1 and increased AIF and calpain1 protein levels in apoptotic cells of preeclamptic placentas. These findings have for the first time revealed that decreased levels of syncytin-1 can trigger the AIF-mediated apoptosis pathway in BeWo cells. This novel mechanism may contribute to the structural and functional deficiencies of syncytium frequently observed in preeclamptic placentas.