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Ling B.,Peking Union Medical College | Zheng H.,Peking Union Medical College | Fu G.,Peking Union Medical College | Yuan J.,Peking Union Medical College | And 10 more authors.
Lung Cancer | Year: 2013

DENND2D was identified as being down-regulated in lung cancer using a lung cancer low-expression suppression subtractive hybridization (SSH) library. In this study, DENND2D down-regulation has been observed not only in non-small cell lung cancer (NSCLC) cell lines and lung squamous cell carcinoma (SCC) tissues, but also in immortalized human bronchial epithelial (IHBE) cell lines and precancerous lesions, indicating that the down-regulation of DENND2D may be an early event in lung cancer. The relative DNA copy number and mRNA and protein expression levels of DENND2D were determined in vitro, and they revealed a complicated regulatory network at the genomic, transcriptional and translational levels. Over-expression of DENND2D significantly suppressed the proliferation of NSCLC cells in vitro and in vivo by inducing apoptosis. These results indicate that DENND2D might function as a tumor suppressor-like gene to prevent the survival and expansion of cells with genetic damage through apoptosis mechanism, and absence of DENND2D might play a permissive role, as an early event, in tumorigenesis. © 2012 Elsevier Ireland Ltd.

Wang P.,Peking University | Wang P.,Chinese National Human Genome Center and 3 707 North YongChang Road | Lu Y.,Chinese National Human Genome Center and 3 707 North YongChang Road | Lu Y.,Dalian Polytechnic University | And 5 more authors.
Life Sciences | Year: 2011

Aims: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has many transcript variants, but whether they possess distinct function is not completely known. In the present study, we compared the function of these TRAIL variants. Main methods: A bioinformatics analysis was performed to examine potential TRAIL variants. For the functional study, over-expression of TRAIL isoforms was used to examine their NF-κB inducing and apoptotic activities in both cancer and normal cells. Moreover, soluble TRAIL E4 variant protein was expressed and purified in prokaryotic cells, and was used for apoptotic assay. Key findings: We cloned seven truncated TRAIL variants, designated as AK, E2, E3, E4, DA, BX424, and BX439. In comparison with the wild type TRAIL protein expressed from full-length RefSeq, over-expression of all these TRAIL variants activated NF-κB and its targeting genes in human cells at varying degrees. Some isoforms including BX424, DA and E4 even showed NF-κB, IL8, CCL4 and CCL20 promoter activating activity stronger than the wild type protein. All truncated variant proteins had no toxicity to normal human cells, similar to the wild type protein; however, they all failed to induce apoptosis in cancer cells that are sensitive to TRAIL. Recombinant soluble TRAIL E4 protein also failed to antagonize TRAIL-induced apoptosis in cancer cells. Significance: Truncated TRAIL variant proteins lost apoptotic activity but retained or even enhanced the NF-κB activating potentials, these results suggest that TRAIL variants may play roles in non-apoptotic cellular processes that are more important than we previously thought. © 2011 Elsevier Inc. All rights reserved.

Peng Z.,Chinese National Human Genome Center and 3 707 North YongChang Road | Peng Z.,Wannan Medical College | Shi T.,Chinese National Human Genome Center and 3 707 North YongChang Road | Shi T.,Peking University | And 2 more authors.
BMC Cell Biology | Year: 2010

Background: RNF122 is a recently discovered RING finger protein that is associated with HEK293T cell viability and is overexpressed in anaplastic thyroid cancer cells. RNF122 owns a RING finger domain in C terminus and transmembrane domain in N terminus. However, the biological mechanism underlying RNF122 action remains unknown.Results: In this study, we characterized RNF122 both biochemically and intracellularly in order to gain an understanding of its biological role. RNF122 was identified as a new ubiquitin ligase that can ubiquitinate itself and undergoes degradation in a RING finger-dependent manner. From a yeast two-hybrid screen, we identified calcium-modulating cyclophilin ligand (CAML) as an RNF122-interacting protein. To examine the interaction between CAML and RNF122, we performed co-immunoprecipitation and colocalization experiments using intact cells. What is more, we found that CAML is not a substrate of ubiquitin ligase RNF122, but that, instead, it stabilizes RNF122.Conclusions: RNF122 can be characterized as a C3H2C3-type RING finger-containing E3 ubiquitin ligase localized to the ER. RNF122 promotes its own degradation in a RING finger-and proteasome-dependent manner. RNF122 interacts with CAML, and its E3 ubiquitin ligase activity was noted to be dependent on the RING finger domain. © 2010 Peng et al; licensee BioMed Central Ltd.

Wang P.,Chinese National Human Genome Center and 3 707 North YongChang Road | Wang P.,Peking University | Sun B.,Tangshan College | Hao D.,Tangshan College | And 5 more authors.
Biochemical and Biophysical Research Communications | Year: 2010

Mitogen-activated protein kinase (MAPK) cascades play an important role in regulation of AP-1 activity through the phosphorylation of distinct substrates. In the present study, we identified a novel protein, TMEM174, whose RNA transcripts are highly expressed in human kidney tissue. TMEM174 is comprised of 243 amino acids, and contains two predicted transmembrane helices which determine its subcellular localization in endoplasmic reticulum and influences its functions. Over-expression of TMME174 enhanced the transcriptional activity of AP-1 and promoted cell proliferation, whereas the truncated mutant TMEM174ΔTM without the transmembrane regions did not retain these functions. The possible mechanism of activation of AP-1 by TMEM174 was further examined. Our results suggest the potential role of TMEM174 in renal development and physiological function. © 2010 Elsevier Inc. All rights reserved.

Shi T.,Chinese National Human Genome Center and 3 707 North YongChang Road | Shi T.,Peking University | Dong Y.,Chinese National Human Genome Center and 3 707 North YongChang Road | Dong Y.,Southern Medical University | And 5 more authors.
Life Sciences | Year: 2010

Aims: Tumor hypoxia is a common phenomenon and hypoxia-inducible factor-1 is the transcription factor that is most closely associated with hypoxia. Hypoxia-inducible factor-1 is overexpressed in most solid tumors and plays a vital role in hypoxic acclimatization, energy metabolism, tumor angiogenesis, tumor invasion, and drug tolerance in cancer cells. We aimed to identify novel human genes associated with the stability and transcriptional activity of hypoxia-inducible factor-1. Main methods: A cell-based dual luciferase reporter system based on a hypoxia responsive element luciferase reporter gene was constructed to screen 409 novel human genes cloned in our lab. Western blot analysis was used to examine the changes in the expression level of hypoxia-inducible factor-1 α, and RT-PCR analysis was used to detect the transcription level of adenylate kinase 3. Key findings: Our results demonstrated that chromatin-modifying protein 4A could significantly up-regulate the hypoxia responsive element luciferase activity under both normoxic and cobalt chloride-induced hypoxic environment in HeLa cells. Moreover, Chromatin-modifying protein 4A could increase the expression of hypoxia-inducible factor-1 α protein under normoxic condition, and enhance the transcription level of adenylate kinase 3, which is one of the target genes of hypoxia-inducible factor-1. Significance: The functional screening platform therefore can be applied for the high-throughput screening of hypoxia-inducible factor-1-related genes, which would provide new insights into underlying molecular mechanisms that may regulate hypoxia in mammalian cells. © 2010 Elsevier Inc.

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