Short interfering RNA against the PDCD5 attenuates cell apoptosis and caspase-3 activity induced by Bax overexpression

The programmed cell death 5 (PDCD5) protein plays an important apoptosis-accelerating role in cells undergoing apoptosis. Decreased expression of PDCD5 has been detected in various human carcinomas. Here we describe that one potent short interfering RNA (siRNA) against the PDCD5 (siPDCD5) specifically inhibits the expression of PDCD5 at both the mRNA and protein level. Cells with decreased PDCD5 expression displayed reduced sensitivity to an apoptotic stimulus induced by Bax overexpression in HeLa, HEK293 and 293T cell lines. Furthermore, we also show that siPDCD5 inhibited both caspase-3 activity and procaspase-3 cleavage. Suppressed expression of PDCD5 attenuates the release of cytochrome c from mitochondria to cytosol induced by Bax overexpression. This phenomenon is accompanied by the reduced translocation of Bax from the cytosol to mitochondria. MTT assay shows that targeted suppression of PDCD5 expression markedly promoted cell proliferation in Hela and HEK293 cell lines. Our data suggests that PDCD5 may exert its effects through pathway of mitochondria by modulating Bax translocation, cytochrome c release and caspase 3 activation directly or indirectly, and that decreased PDCD5 expression may be one of the mechanisms by which tumor cells achieve resistance to apoptotic stimulus induced by anticancer drugs.     

Effects of Wnt proteins on cell proliferation and apoptosis in HEK293 cells

Wnt proteins and Wnt signalings have been implicated in a variety of development and cell processes, while aberrant activation of Wnt signaling is linked to a range of cancers in many tissues. In this study, we used the HEK293 cell line to investigate the effects of Wnt3a and Wnt5a on proliferation and apoptosis in a serum starvation culture. After Wnt3a and Wnt5a proteins were expressed, they both promoted the proliferation of HEK293 cells under serum starvation. After 48h of serum starvation, both Wnt3a and Wnt5a inhibited serum starvation-induced apoptosis of HEK293 cells and continued up to 96h. We demonstrated that Wnt3a and Wnt5a can promote proliferation of HEK293 cells and inhibit serum starvation-induced apoptosis, which implies that Wnt3a and Wnt5a can maintain the survival of HEK293 cells under stress, and also provide a novel insight into the role of Wnt3a and Wnt5a and their related signalings in carcinogenesis.     

Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIP(L) in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.     

外源过表达CPNE5基因增强HEK293细胞对Zeocin的敏感性

目的：探讨Zeocin处理是否能增强CPNE5基因诱导细胞凋亡的作用。方法：分别构建CPNE5基因稳定过表达和抑表达载体,转染HEK293细胞,形成克隆后,用RT-PCR、MTT法分析CPNE5基因过表达和抑表达的HEK293单克隆细胞对Zeocin作用的敏感性。结果：CPNE5基因过表达的HEK293单克隆细胞对Zeocin反应敏感性增加。结论：Zeocin能增强CPNE5基因诱导细胞凋亡的作用。     

镉诱导HEK293细胞凋亡及其线粒体凋亡途径

本课题研究了氯化镉(CdCl_2)诱导HEK293细胞(人胚胎肾细胞系)的凋亡,初步探讨了凋亡过程中Caspase-3、Bcl-2的变化和凋亡诱导因子(AIF)的转移以及它们的意义。MTT法检测CdCl_2对HEK293细胞增殖的抑制作用;通过倒置显微镜、电镜、琼脂糖凝胶电泳、流式细胞术、激光共聚焦观察细胞凋亡;应用Western blot法和荧光免疫法测定Caspase-3酶原、Bcl-2蛋白的变化以及检测AIF蛋白在细胞中的定位。结果显示:CdCl_2对HEK293细胞具有显著的生长抑制作用,并呈明显的剂量和时间依赖性。在琼脂糖凝胶电泳中,显示有凋亡细胞特有的DNA梯状条带,其中30μmol/L作用6-9h梯状条带最为清晰,时间过长或浓度过高则梯状条带逐渐模糊,表明镉浓度过高或处理时间过长,细胞有坏死。流式细胞仪检测也印证了这一结果。形态学观察可见明显的细胞凋亡特征。同时线粒体膜电位明显下降,发现Caspase-3酶原蛋白、Bcl-2蛋白含量减少,并具有时间依赖性;另外检测到线粒体AIF向细胞核转移。而Bcl-2转染后有一定的抑制凋亡作用。实验结果提示,CdCl_2能够诱导HEK293细胞凋亡,线粒体损伤导致AIF转移与细胞色素c释放,从而引发的非Caspases与Caspases凋亡途径可能在镉引发的细胞凋亡过程中起重要作用,而Caspase-3, Bcl-2起着重要的调控作用。     

Unraveling molecular effects of ADAR1 overexpression in HEK293T cells by label-free quantitative proteomics

ADAR1 is a double-stranded RNA (dsRNA) editing enzyme that specifically converts adenosine to inosine. ADAR1 is ubiquitously expressed in eukaryotes and participate in various cellular processes such as differentiation, proliferation and immune responses. We report here a new proteomics study of HEK293T cells with and without ADAR1 overexpression. The up- and down-regulated proteins by ADAR1 overexpression are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by label-free protein quantification. Totally 1,495 proteins (FDR < 0.01) are identified, among which 211 are up- and 159 are down-regulated for at least 1.5-fold (n = 3, p < 0.05). Gene ontology analysis reveals that these ADAR1-regulated proteins are involved in protein translation and cell cycle regulation. Bioinformatics analysis identifies a closely related network consistent for the protein translation machinery and a tightly connected network through proliferating cell nuclear antigen (PCNA)-interactions. Up-regulation of the proteins in the PCNA-mediated cell proliferation network is confirmed by Western blotting. In addition, ADAR1 overexpression is confirmed to increase cell proliferation in HEK293T cells and A549 cells. We conclude that ADAR1 overexpression modulates the protein translation and cell cycle networks through PCNA-mediated protein-protein interaction to promote cell proliferation in HEK293 cells.     

Knockdown of KDM2A inhibits proliferation associated with TGF-β expression in HEK293T cell

Lysine-specific demethylase 2A (KDM2A, also known as JHDM1A or FBXL11) plays an important role in regulating cell proliferation. However, the mechanisms on KDM2A controlling cell proliferation are varied among cell types, even controversial conclusions have been drawn. In order to elucidate the functions and underlying mechanisms for KDM2A controlling cell proliferation and apoptosis, we screened a KDM2A knockout HEK293T cell lines by CRISPR–Cas9 to illustrate the effects of KDM2A on both biological process. The results indicate that knocking down expression of KDM2A can significantly weaken HEK293T cell proliferation. The cell cycle analysis via flow cytometry demonstrate that knockdown expression of KDM2A will lead more cells arrested at G2/M phase. Through the RNA-seq analysis of the differential expressed genes between KDM2A knockdown HEK293T cells and wild type, we screened out that TGF-β pathway was significantly downregulated in KDM2A knockdown cells, which indicates that TGF-β signaling pathway might be the downstream target of KDM2A to regulate cell proliferation. When the KDM2A knockdown HEK293T cells were transient-transfected with KDM2A overexpression plasmid or treated by TGF-β agonist hydrochloride, the cell proliferation levels can be partial or completely rescued. However, the TGF-β inhibitor LY2109761 can significantly inhibit the KDM2A WT cells proliferation, but not the KDM2A knockdown HEK293T cells. Taken together, these findings suggested that KDM2A might be a key regulator of cell proliferation and cell cycle via impacting TGF-β signaling pathway.

     

Ectopic Expression of MmKin17 Protein Inhibits Cell Proliferation of Human Tumor-Derived Cells

To characterize the biological role of Kin17 protein, a mammalian nuclear protein which participates in the response to UV and ionizing radiation and binds to curved DNA, EBV-derived vectors carrying (Mm)Kin17 cDNA were constructed and transfected in tumorigenic cells harboring different p53 profiles (HeLa, H1299, and HCT116) and in immortalized HEK 293 cells. (Mm)Kin17 protein expression induced a tremendous decrease in cell proliferation of the three tumorigenic cell lines 2 weeks after transfection. Transfection of HEK 293 cells with an pEBVCMV(Mm)Kin17 plasmid gave rise to numerous (Mm)Kin17-expressing cells which constantly disappeared with time, preventing the establishment of (Mm)Kin17-expressing cells. Several independent clones were isolated from HEK 293 cells carrying a pEBVMT(Mm)Kin17 vector. The two clones described here (B223.1 and B223.2) exhibited different (Mm)Kin17 protein levels and displayed a gradual decrease in their proliferative capacities. In B223.1 cells, the basal expression of (Mm)Kin17 greatly reduced plating efficiency and cell growth. B223.1 cell morphology was altered, with numerous round-shaped cells whose spreading on the culture support was hampered. We observed giant multinucleated cells or cells containing micronuclei-like structures and/or multilobed nuclei. To conclude, (Mm)Kin17 overexpression reduced the proliferation of tumorigenic cells independently of their p53 status and modified cell growth and cell morphology of established HEK 293 cells producing (Mm)Kin17 protein. It is likely that (Mm)Kin17 may interfere with DNA replication.     

早老素通过下调CDK4使HEK293T细胞周期阻滞

早老素（progerin）的累积导致儿童早老症（Hutchinson Gilford progeria syndrome, HGPS）的发生,并与正常衰老相关。早老素能使细胞内稳态失衡但分子机制仍有待深入研究。本研究旨在探讨早老素导入人胚胎肾293T细胞（human embryo kidney 293T cell, HEK293T）后细胞增殖、周期变化的分子机制。形态学观察发现过表达早老素的HEK293T细胞密度下降,(57±2.47)%细胞核形态皱缩。细胞增殖和周期实验证明早老素使细胞增殖减慢,发生G1/S期阻滞,G1细胞从 (42.3±1.31)%升至(47.2±1.26)%,而S期细胞从 (43.1±1.36)%降至 (38.5±1.42)%。Western印迹结果显示早老素的高表达引起p21蛋白表达上调（103.2±1.49）%,CDK4下调（63±1.52）%,而p53、ATM、CyclinE1以及p16等蛋白质水平均不变;HEK293T细胞中早老素的过表达导致γ H2AX水平下调（53±1.36）%,H2O2处理后变化趋势不变。我们的研究结果提示,早老素通过上调p21和下调CDK4使细胞发生周期阻滞,不能增加HEK293T细胞的损伤及衰老。     

Overexpression of thioredoxin reductase 1 inhibits migration of HEK-293 cells

Background information. TrxR (thioredoxin reductase), in addition to protecting against oxidative stress, plays a role in the redox regulation of intracellular signalling pathways controlling, among others, cell proliferation and apoptosis. The aim of the present study was to determine whether TrxR1 is involved in the regulation of cell migration. Results. Stably transfected HEK‐293 (human embryonic kidney) cells which overexpress cytosolic TrxR1 (HEK‐TrxR15 and HEK‐TrxR11 cells) were used in the present study. We found that the stimulation of cell motility induced by PKC (protein kinase C) activators, PMA and DPhT (diphenyltin), was inhibited significantly in the HEK‐TrxR15 and HEK‐TrxR11 cells compared with control cells. The overexpression of TrxR1 also inhibited characteristic morphological changes and reorganization of the F‐actin cytoskeleton induced by PMA and DPhT. In addition, the selective activation of PKCδ by DPhT was inhibited in cells that overexpressed cytosolic TrxR1. Furthermore, rottlerin, a selective inhibitor of PKCδ, and PKCδ siRNA (small interfering RNA), suppressed the morphological changes induced by DPhT in the control cells. Conclusions. The overexpression of TrxR1 inhibits migration of HEK‐293 cells stimulated with PMA and DPhT. Moreover, our observations suggest that this effect is mediated by the inhibition of PKCδ activation.     

Overexpression of Nrdp1/FLRF sensitizes cells to oxidative stress

Nrdp1 is a RING finger containing ubiquitin E3 ligase that interacts with and modulates activity of multiple proteins, including ErbB3 and Parkin, a causative protein for early onset recessive juvenile parkinsonism (AR-JP). To investigate the functions of Nrdp1, we have generated stable Tet-On inducible HEK293 cells that overexpress Flag-tagged full length Nrdp1, N-terminal Nrdp1 and C-terminal Nrdp1. We demonstrate that overexpression of full-length Nrdp1, not Nrdp1 N-terminus or Nrdp1 C-terminus in cultured HEK293 cells, inhibits cell growth. In addition, we have treated cells with hydroxynonenal (HNE), 6-hydroxydopamine (6-OHDA), and hydrogen peroxide (H₂O₂) at different concentrations. We have found that Nrdp1 overexpression sensitizes HEK293 cells to oxidative stressors in a dosage-dependent manner. Our data provide insights into understanding the potential role of Nrdp1 in cell growth, apoptosis and oxidative stress, and in the pathogenesis of Parkinson’s disease.     

PTEN基因诱导人胚肾293细胞凋亡和细胞周期停滞

为了研究抑癌基因PTEN过表达对HEK293细胞凋亡和细胞周期停滞的作用,以野生型PTEN和PTEN突变子(T910G)表达质粒分别转染无PTEN表达的人胚肾293细胞,采用细胞质梯度DNA方法检测细胞凋亡,以流式细胞仪分析细胞周期.发现PTEN过表达能够诱导人胚肾293细胞质中出现梯度DNA,293细胞发生凋亡,PTEN过表达改变细胞周期分布,G0/G1期细胞增加13%,S期细胞下降15%.PTEN突变子对细胞凋亡和G1细胞停滞的影响略弱于野生型PTEN.PTEN基因过表达明显下调血小板衍生生长因子(PDGF)诱导的蛋白激酶B(PKB)和p42,p44-促分裂原活化蛋白激酶(MAPK)磷酸化水平,PTEN突变子对p42,p44-MAPK磷酸化水平的调节作用略弱于野生型PTEN.PTEN通过抑制细胞增殖,诱导细胞凋亡而影响细胞生长.     

Ectopic expression of Flt3 kinase inhibits proliferation and promotes cell death in different human cancer cell lines

Stable ectopic expression of Flt3 receptor tyrosine kinase is usually performed in interleukin 3 (IL-3)-dependent murine cell lines like Ba/F3, resulting in loss of IL-3 dependence. Such high-level Flt3 expression has to date not been reported in human acute myeloid leukemia (AML) cell lines, despite the fact that oncogenic Flt3 aberrancies are frequent in AML patients. We show here that ectopic Flt3 expression in different human cancer cell lines might reduce proliferation and induce apoptotic cell death, involving Bax/Bcl2 modulation. Selective depletion of Flt3-expressing cells occurred in human AML cell lines transduced with retroviral Flt3 constructs, shown here using the HL-60 leukemic cell line. Flt3 expression was investigated in two cellular model systems, the SAOS-2 osteosarcoma cell line and the human embryonic kidney HEK293 cell line, and proliferation was reduced in both systems. HEK293 cells underwent apoptosis upon ectopic Flt3 expression and cell death could be rescued by overexpression of Bcl-2. Furthermore, we observed that the Flt3-induced inhibition of proliferation in HL-60 cells appeared to be Bax-dependent. Our results thus suggest that excessive Flt3 expression has growth-suppressive properties in several human cancer cell lines.     

High glucose alters apoptosis and proliferation in HEK293 cells by inhibition of cloned BK Ca channel

It has been reported that diabetic vascular dysfunction is associated with impaired function of large conductance Ca(2+) -activated K(+) (BK(Ca) ) channels. However, it is unclear whether impaired BK(Ca) channel directly participates in regulating diabetic vascular remodeling by altering cell growth in response to hyperglycemia. In the present study, we investigated the specific role of BK(Ca) channel in controlling apoptosis and proliferation under high glucose concentration (25 mM). The cDNA encoding the α+β1 subunit of BK(Ca) channel, hSloα+β1, was transiently transfected into human embryonic kidney 293 (HEK293) cells. Cloned BK(Ca) currents were recorded by both whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry and analysis of fragmented DNA by agarose gel electrophoresis. Cell proliferation was investigated by flow cytometry assays, MTT test, and immunocytochemistry. In addition, the expression of anti-apoptotic protein Bcl-2, intracellular Ca(2+) , and mitochondrial membrane potential (Δψm) were also examined to investigate the possible mechanisms. Our results indicate that inhibition of cloned BK(Ca) channels might be responsible for hyperglycemia-altered apoptosis and proliferation in HEK-hSloα+β1 cells. However, activation of BK(Ca) channel by NS1619 or Tamoxifen significantly induced apoptosis and suppressed proliferation in HEK-hSloα+β1 cells under hyperglycemia condition. When rat cerebral smooth muscle cells were cultured in hyperglycemia, similar findings were observed. Moreover, the possible mechanisms underlying the activation of BK(Ca) channel were associated with decreased expression of Bcl-2, elevation of intracellular Ca(2+) , and a concomitant depolarization of Δψm in HEK-hSloα+β1 cells. In conclusion, cloned BK(Ca) channel directly regulated apoptosis and proliferation of HEK293 cell under hyperglycemia condition.     

Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells

Inactivation of p53 protein by endogenous and exogenous carcinogens is involved in the pathogenesis of different human malignancies. In cancer associated with SV-40 DNA tumor virus, p53 is considered to be non-functional mainly due to its interaction with the large T-antigen. Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in FK866-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53.     

Sphingosine-phosphate lyase enhances stress-induced ceramide generation and apoptosis

Sphingosine-1-phosphate lyase is a widely expressed enzyme that catalyzes the essentially irreversible cleavage of the signaling molecule sphingosine 1-phosphate. To investigate whether sphingosine-1-phosphate lyase influences mammalian cell fate decisions, a recombinant human sphingosine-1-phosphate lyase fused to green fluorescent protein was expressed in HEK293 cells. The recombinant enzyme was active, localized to the endoplasmic reticulum, and reduced baseline sphingosine and sphingosine 1-phosphate levels. Stable overexpression led to diminished viability under stress, which was attributed to an increase in apoptosis and was reversible in a dose-dependent manner by exogenous sphingosine 1-phosphate. In contrast to sphingosine 1-phosphate, the products of the lyase reaction had no effect on apoptosis. Lyase enzymatic activity was required to potentiate apoptosis, because cells expressing a catalytically inactive enzyme behaved like controls. Stress increased the amounts of long- and very long-chain ceramides in HEK293 cells, and this was enhanced in cells overexpressing wild type but not catalytically inactive lyase. The ceramide increases appeared to be required for apoptosis, because inhibition of ceramide synthase with fumonisin B1 decreased apoptosis in lyase-overexpressing cells. Thus, sphingosine-1-phosphate lyase overexpression in HEK293 cells decreases sphingosine and sphingosine 1-phosphate amounts but elevates stress-induced ceramide generation and apoptosis. This identifies sphingosine-1-phosphate lyase as a dual modulator of sphingosine 1-phosphate and ceramide metabolism as well as a regulator of cell fate decisions and, hence, a potential target for diseases with an imbalance in these biomodulators, such as cancer.     

Human TRPV5 and TRPV6: Key players in cadmium and zinc toxicity

TRPV5 and TRPV6 are two major calcium transport pathways in the human body maintaining calcium homeostasis. TRPV5 is mainly expressed in the distal convoluted and connecting tubule where it is the major, regulated pathway for calcium reabsorption. TRPV6 serves as an important calcium entry pathway in the duodenum and the placenta. Previously, we showed that human TRPV6 (hTRPV6) transports several heavy metals. In this study we tested whether human TRPV5 (hTRPV5) also transports cadmium and zinc, and whether hTRPV5 together with hTRPV6 are involved in cadmium and zinc toxicity. The hTRPV5 mRNA and protein were expressed in HEK293 cells transiently transfected with pTagRFP-C1-hTRPV5. The overexpression of the hTRPV5 protein at the plasma membrane was revealed by cell surface biotinylation and immunofluorescence techniques. We observed that both cadmium and zinc permeate hTRPV5 in ion imaging experiments using Fura-2 or Newport Green DCF. Our results were further confirmed using whole-cell patch clamp technique. Transient overexpression of hTRPV5 or hTRPV6 sensitized cells to cadmium and zinc. Toxicity curves of cadmium and zinc were also shifted in hTRPV6 expressing HEK293 cells clones. Our results suggest that TRPV5 and TRPV6 are crucial gates controlling cadmium and zinc levels in the human body especially under low calcium dietary conditions, when these channels are maximally upregulated.     

Involvement of N-terminal-extended form of sphingosine kinase 2 in serum-dependent regulation of cell proliferation and apoptosis

Sphingosine kinase (SPHK) 1 is implicated in the regulation of cell proliferation and anti-apoptotic processes by catalyzing the formation of an important bioactive messenger, sphingosine 1-phosphate. Unlike the proliferative action of SPHK1, another isozyme, SPHK2, has been shown to possess anti-proliferative or pro-apoptotic action. Molecular mechanisms of SPHK2 action, however, are largely unknown. The present studies were undertaken to characterize the N-terminal-extended form of SPHK2 (SPHK2-L) by comparing it with the originally reported form, SPHK2-S. Real-time quantitative PCR analysis revealed that SPHK2-L mRNA is the major form in several human cell lines and tissues. From sequence analyses it was concluded that SPHK2-L is a species-specific isoform that is expressed in human but not in mouse. At the protein level it has been demonstrated by immunoprecipitation studies that SPHK2-L is the major isoform in human hepatoma HepG2 cells. SPHK2-L, when expressed in human embryonic kidney (HEK) 293 cells, did not show any inhibition of DNA synthesis in the presence of serum, whereas it showed marked inhibition in the absence of serum. Moreover, serum deprivation resulted in the translocation of SPHK2-L into the nuclei. In addition, serum deprivation induced SPHK2-L expression in HEK293 cells. Furthermore, suppression of SPHK2 by small interfering RNA treatment prevented serum deprivation- or drug-induced apoptosis in HEK293 cells. Taken together, these results indicate that a major form of SPHK2 splice variant, SPHK2-L, in human cells does not inhibit DNA synthesis under normal conditions and that SPHK2-L accumulation in the nucleus induced by serum deprivation may be involved in the cessation of cell proliferation or apoptosis depending on the cell type.