High-throughput RNAi screening by time-lapse imaging of live human cells

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.     

Depletion of the nucleolar protein nucleostemin causes G1 cell cycle arrest via the p53 pathway

Nucleostemin (NS) is a nucleolar protein expressed in adult and embryo-derived stem cells, transformed cell lines, and tumors. NS decreases when proliferating cells exit the cell cycle, but it is unknown how NS is controlled, and how it participates in cell growth regulation. Here, we show that NS is down-regulated by the tumor suppressor p14(ARF) and that NS knockdown elevates the level of tumor suppressor p53. NS knockdown led to G1 cell cycle arrest in p53-positive cells but not in cells in which p53 was genetically deficient or depleted by small interfering RNA knockdown. These results demonstrate that, in the cells investigated, the level of NS is regulated by p14(ARF) and the control of the G1/S transition by NS operates in a p53-dependent manner.     

Inhibition of cell division by the human cytomegalovirus IE86 protein: role of the p53 pathway or cyclin-dependent kinase 1/cyclin B1

The human cytomegalovirus (HCMV) IE86 protein induces the human fibroblast cell cycle from G(0)/G(1) to G(1)/S, where cell cycle progression stops. Cells with a wild-type, mutated, or null p53 or cells with null p21 protein were transduced with replication-deficient adenoviruses expressing HCMV IE86 protein or cellular p53 or p21. Even though S-phase genes were activated in a p53 wild-type cell, IE86 protein also induced phospho-Ser(15) p53 and p21 independent of p14ARF but dependent on ATM kinase. These cells did not enter the S phase. In human p53 mutant, p53 null, or p21 null cells, IE86 protein did not up-regulate p21, cellular DNA synthesis was not inhibited, but cell division was inhibited. Cells accumulated in the G(2)/M phase, and there was increased cyclin-dependent kinase 1/cyclin B1 activity. Although the HCMV IE86 protein increases cellular E2F activity, it also blocks cell division in both p53(+/+) and p53(-/-) cells.     

Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.     

LYG-202 inhibits the proliferation of human colorectal carcinoma HCT-116 cells through induction of G1/S cell cycle arrest and apoptosis via p53 and p21(WAF1/Cip1) expression

We recently established that LYG-202, a new flavonoid with a piperazine substitution, exerts an anti-tumor effect in vivo and in vitro. In the present study, we demonstrate that LYG-202 induces G1/S phase arrest and apoptosis in human colorectal carcinoma HCT-116 cells. Data showed that the blockade of the cell cycle was associated with increased p21(WAF1/Cip1) and Rb levels and reduced expression of cyclin D1, cyclin E, and CDK4. Moreover, PARP cleavage, activation of caspase-3, caspase-8, and caspase-9, and an increased ratio of Bax/Bcl-2 were detected in LYG-202-induced apoptosis. Additionally, activation of p53 resulted in the up-regulation of its downstream targets PUMA and p21(WAF1/Cip1), as well as the down-regulation of its negative regulator MDM2, suggesting that the p53 pathway may play a crucial role in LYG-202-induced cell cycle arrest and apoptosis. Furthermore, siRNA knockdown of p53 attenuated the G1 cell cycle arrest and apoptosis induced by LYG-202, as the effects of LYG-202 on up-regulation of p21(WAF1/Cip1) and down-regulation of Bcl-2 and pro-caspase-3 were partly inhibited in p53 siRNA transfected cells compared with control siRNA transfected cells. Collectively, these data indicate that LYG-202 exerts its anti-tumor potency by activating the p53-p21 pathway for G1/S cell cycle arrest and apoptosis in colorectal cancer cells.     

Zinc-induced G2/M blockage is p53 and p21 dependent in normal human bronchial epithelial cells

The involvement of p53 and p21 signal pathway in the G2/M cell cycle progression of zinc-supplemented normal human bronchial epithelial (NHBE) cells was examined using the small interferring RNA (siRNA) approach. Cells were cultured for one passage in a different concentration of zinc: <0.4 microM (ZD) as zinc deficient; 4 microM as normal zinc level (ZN) in culture medium; 16 microM (ZA) as normal human plasma zinc level; and 32 microM (ZS) as the high end of plasma zinc attainable by oral supplementation. Nuclear p21 protein and mRNA levels as well as promoter activity in ZS cells, but not in ZD cells, were markedly elevated to almost twofold compared with ZN control cells. G2/M blockage in ZS cells was coupled with the observation of elevated p21 gene expression. In ZS cells, the abrogation of p21 protein induction by the transfection of p21 siRNA was shown to alleviate the G2/M blockage, demonstrating the positive linkage of p21 elevation and G2/M blockage. Abolishment of the increase in p53 protein in ZS cells with transfection of p53 siRNA normalized the elevated p21 protein to a similar level as in ZN control cells, which demonstrated that the p21 induction is p53 dependent. Furthermore, the normalization of p53 protein by siRNA treatment in ZS cells alleviated cell growth depression and G2/M blockage, which demonstrated that p53 was involved in the high zinc status-induced G2/M blockage and growth depression. Thus high zinc status in NHBE cells upregulates p53 expression which in turn elevates p21 that eventually induces G2/M blockage.     

A genome-wide RNAi screen identifies novel targets of neratinib sensitivity leading to neratinib and paclitaxel combination drug treatments

ErbB2 is frequently activated in tumors, and influences a wide array of cellular functions, including proliferation, apoptosis, cell motility and adhesion. HKI-272 (neratinib) is a small molecule pan-kinase inhibitor of the ErbB family of receptor tyrosine kinases, and shows strong antiproliferative activity in ErbB2-overexpressing breast cancer cells. We undertook a genome-wide pooled lentiviral RNAi screen to identify synthetic lethal or enhancer (synthetic modulator screen) genes that interact with neratinib in a human breast cancer cell line (SKBR-3). These genes upon knockdown would modulate cell viability in the presence of subeffective concentrations of neratinib. We discovered a diverse set of genes whose depletion selectively impaired or enhanced the viability of SKBR-3 cells in the presence of neratinib. We observed diverse pathways including EGFR, hypoxia, cAMP, and protein ubiquitination that, when co-treated with RNAi and neratinib, resulted in arrest of cell proliferation. Examining the changes of these genes and their protein products also led to a rationale for clinically relevant drug combination treatments. Treatment of cells with either paclitaxel or cytarabine in combination with neratinib resulted in a strong antiproliferative effect. The identification of novel mediators of cellular response to neratinib and the development of potential drug combination treatments have expanded our understanding of neratinib's mode-of-action for the development of more effective therapeutic regimens. Notably, our findings support a paclitaxel and neratinib phase III clinical trial in breast cancer patients.     

Development of a high-content screening assay panel to accelerate mechanism of action studies for oncology research

Efficient elucidation of the biological mechanism of action of novel compounds remains a major bottleneck in the drug discovery process. To address this need in the area of oncology, we report the development of a multiparametric high-content screening assay panel at the level of single cells to dramatically accelerate understanding the mechanism of action of cell growth-inhibiting compounds on a large scale. Our approach is based on measuring 10 established end points associated with mitochondrial apoptosis, cell cycle disruption, DNA damage, and cellular morphological changes in the same experiment, across three multiparametric assays. The data from all of the measurements taken together are expected to help increase our current understanding of target protein functions, constrain the list of possible targets for compounds identified using phenotypic screens, and identify off-target effects. We have also developed novel data visualization and phenotypic classification approaches for detailed interpretation of individual compound effects and navigation of large collections of multiparametric cellular responses. We expect this general approach to be valuable for drug discovery across multiple therapeutic areas.     

Kruppel-like factor 4 mediates p53-dependent G1/S cell cycle arrest in response to DNA damage

The tumor suppressor p53 is required for the maintenance of genomic integrity following DNA damage. One mechanism by which p53 functions is to induce a block in the transition between the G(1) and S phase of the cell cycle. Previous studies indicate that the Krüppel-like factor 4 (KLF4) gene is activated following DNA damage and that such activation depends on p53. In addition, enforced expression of KLF4 causes G(1)/S arrest. The present study examines the requirement of KLF4 in mediating the p53-dependent cell cycle arrest process in response to DNA damage. We show that the G(1) population of a colon cancer cell line, HCT116, that is null for the p53 alleles (-/-) was abolished following gamma irradiation compared with cells with wild-type p53 (+/+). Conditional expression of KLF4 in irradiated HCT116 p53-/- cells restored the G(1) cell population to a level similar to that seen in irradiated HCT116 p53+/+ cells. Conversely, treatment of HCT116 p53+/+ cells with small interfering RNA (siRNA) specific for KLF4 significantly reduced the number of cells in the G(1) phase following gamma irradiation compared with the untreated control or those treated with a nonspecific siRNA. In each case the increase or decrease in KLF4 level because of conditional induction or siRNA inhibition, respectively, was accompanied by an increase or decrease in the level of p21(WAF1/CIP1). Results of our study indicate that KLF4 is an essential mediator of p53 in controlling G(1)/S progression of the cell cycle following DNA damage.     

Selenoprotein W depletion induces a p53- and p21-dependent delay in cell cycle progression in RWPE-1 prostate epithelial cells

The anticancer activity of selenium (Se) has been demonstrated in myriad animal and in vitro studies, yet the mechanisms remain obscure. The main form of Se in animal tissues is selenocysteine in selenoproteins, but the relative importance of selenoproteins versus smaller Se compounds in cancer protection is unresolved. Selenoprotein W (SEPW1) is a highly conserved protein ubiquitously expressed in animals, bacteria, and archaea. SEPW1 depletion causes a delay in cell cycle progression at the G1/S transition of the cell cycle in breast and prostate epithelial cells. Tumor suppressor protein p53 is a master regulator of cell cycle progression and is the most frequently mutated gene in human cancers. p53 was increased in SEPW1 silenced cells and was inversely correlated with SEPW1 mRNA in cell lines with altered SEPW1 expression. Silencing SEPW1 decreased ubiquitination of p53 and increased p53 half-life. SEPW1 silencing increased p21(Cip1/WAF1/CDKN1A), while p27 (Kip1/CDKN1B) levels were unaffected. G1-phase arrest from SEPW1 knockdown was abolished by silencing p53 or p21. Cell cycle arrest from SEPW1 silencing was not associated with activation of ATM or phosphorylation of Ser-15 in p53, suggesting the DNA damage response pathway was not involved. Silencing GPX1 had no effect on cell cycle, suggesting that G1-phase arrest from SEPW1 silencing was not due to loss of antioxidant protection. More research is required to identify the function of SEPW1 and how it affects stability of p53.     

siRNA干扰Mad2蛋白的表达对乳腺癌MCF-7细胞紫杉醇药物敏感性的影响

目的研究纺锤体检测点蛋白Mad2对乳腺癌细胞MCF-7紫杉醇敏感性的影响。方法运用小片段干扰RNA技术下调MCF-7细胞中Mad2蛋白的表达,经不同浓度的紫杉醇药物浓度处理,通过细胞术检测细胞周期分布,MTY法检测细胞存活率的变化,RT—PCR及Western印迹法检测相关蛋白的表达改变情况。结果siRNA可以特异性干扰MCF-7细胞中Mad2蛋白水平的表达,同时显著降低紫杉醇诱导细胞发生有丝分裂阻滞的比例,导致CyclinB1的表达下调,凋亡率降低;并且Mad2表达下调后,MCF-7细胞中黏附分子E-cadherin的表达量也降低。结论Mad2在紫杉醇诱导MCF-7细胞发生有丝分裂周期阻滞中发挥重要作用,进而影响细胞对紫杉醇的敏感性,同时细胞黏附分子E—cadherin可能参与这一过程的发生。     

Cell Proliferation and Motility Are Inhibited by G1 Phase Arrest in 15-kDa Selenoprotein-Deficient Chang Liver Cells

The 15-kDa selenoprotein (Sep15) is a selenoprotein residing in the lumen of the endoplasmic reticulum (ER) and implicated in quality control of protein folding. Herein, we established an inducible RNAi cell line that targets Sep15 mRNA in Chang liver cells. RNAi-induced Sep15 deficiency led to inhibition of cell proliferation, whereas cell growth was resumed after removal of the knockdown inducer. Sep15-deficient cells were arrested at the G1 phase by upregulating p21 and p27, and these cells were also characterized by ER stress. In addition, Sep15 deficiency led to the relocation of focal adhesions to the periphery of the cell basement and to the decrease of the migratory and invasive ability. All these changes were reversible depending on Sep15 status. Rescuing the knockdown state by expressing a silent mutant Sep15 mRNA that is resistant to siRNA also reversed the phenotypic changes. Our results suggest that SEP15 plays important roles in the regulation of the G1 phase during the cell cycle as well as in cell motility in Chang liver cells, and that this selenoprotein offers a novel functional link between the cell cycle and cell motility.     

Down-regulation of TSG101 by small interfering RNA inhibits the proliferation of breast cancer cells through the MAPK/ERK signal pathway

We designed to investigate the effects of down-regulating the tumor susceptibility gene 101 (TSG101) on the proliferation and apoptosis of the human breast cancer MCF-7 cell line, and the role of the MAPK/ERK signal pathway in this process. The siRNA against TSG101 was transfected into the breast cancer MCF-7 cell line using Lipofectamine 2000. After TSG101 knockdown, the proliferation of MCF-7 cells was measured by the MTT assay. The cell cycle distribution and apoptosis were examined by using flow cytometry while cell migration was measured using a transwell assay. The protein level of p-ERK was further assessed by immunofluorescence and western blotting. Our results are as following, the MCF-7 cells transfected with TSG101 siRNA proliferated significantly slower and exhibited significantly increased rates of apoptosis compared to the control cells. In the TSG101 siRNA transfected cells, the percentage of cells in the G?/G? and S phase of the cell cycle was significantly higher and lower, respectively, compared to the control cells. Moreover, the migration ability of TSG101 siRNA transfected cells was lower than the control groups. Lastly, the level of p-ERK protein in TSG101 siRNA transfected cells was significantly decreased compared with the control cells. In conclusion, TSG101 knockdown in breast cancer cells induces apoptosis and inhibits proliferation. The TSG101 depleted cells are arrested at the G?/S transition of the cell cycle. The migration of breast cancer cells is also impaired by TSG101 siRNA. TSG101 may play a biological role through modulation of the MAPK/ERK signaling pathway in breast cancer.     

Mitotic control by mRNA splicing regulators ensures primary cilia formation

The biogenesis of the primary cilium is coordinated with cell cycle exit/re-entry in most types of cells. After serum starvation, the cilia-generating cells enter quiescence and produce the primary cilium; upon re-addition of serum, they re-enter the cell cycle and resorb the cilium. We previously identified novel mechanisms to link cell cycle progression and ciliogenesis by high-content genome-wide RNAi cell-based screening. In the present study, we pay attention to reveal the impact of mRNA splicing on cilia assembly after mitosis of cell cycle. We demonstrate that splicing regulators such as SON and XAB2 play an important role in mitosis exit, and thus affect ciliogenesis in G1/G0 phases. Knockdown of the splicing regulators in hTERT-RPE1 cells caused abnormal G2/M arrest under both serum addition and serum starvation, indicating defects in mitosis exit. Moreover, the knockdown cells failed to assemble the cilia under serum starvation and an inhibition of mRNA splicing using SSA, a spliceosome inhibitor, also revealed ciliogenesis defect. Finally, we show that the SSA-treated zebrafish display abnormal vascular development as a ciliary defect. These findings suggest the pivotal role of mRNA splicing regulators in cilia assembly and underscore the importance of mitotic regulation in ciliogenesis.     

The regulation of p53, p38 MAPK,JNK and XBP-1s by sphingosine kinases in human embryonic kidney cells

Since inhibitors of sphingosine kinases (SK1, SK2) have been shown to induce p53-mediated cell death, we have further investigated their role in regulating p53, stress activated protein kinases and XBP-1s in HEK293T cells. Treatment of these cells with the sphingosine kinase inhibitor, SKi, which fails to induce apoptosis, promoted the conversion of p53 into two proteins with molecular masses of 63 and 90 kDa, and which was enhanced by over-expression of ubiquitin. The SKi induced conversion of p53 to p63/p90 was also enhanced by siRNA knockdown of SK1, but not SK2 or dihydroceramide desaturase (Degs1), suggesting that SK1 is a negative regulator of this process. In contrast, another sphingosine kinase inhibitor, ABC294640 only very weakly stimulated formation of p63/p90 and induced apoptosis of HEK293T cells. We have previously shown that SKi promotes the polyubiquitination of Degs1, and these forms positively regulate p38 MAPK/JNK pathways to promote HEK293T cell survival/growth. siRNA knockdown of SK1 enhanced the activation of p38 MAPK/JNK pathways in response to SKi, suggesting that SK1 functions to oppose these pro-survival pathways in HEK293T cells. SKi also enhanced the stimulatory effect of the proteasome inhibitor, MG132 on the expression of the pro-survival protein XBP-1s and this was reduced by siRNA knockdown of SK2 and increased by knockdown of p53. These findings suggest that SK1 and SK2 have opposing roles in regulating p53-dependent function in HEK293T cells.     

Single cell cytometry of protein function in RNAi treated cells and in native populations

Background

High Content Screening has been shown to improve results of RNAi and other perturbations, however significant intra-sample heterogeneity is common and can complicate some analyses. Single cell cytometry can extract important information from subpopulations within these samples. Such approaches are important for immune cells analyzed by flow cytometry, but have not been broadly available for adherent cells that are critical to the study of solid-tumor cancers and other disease models.

Results

We have directly quantitated the effect of resolving RNAi treatments at the single cell level in experimental systems for both exogenous and endogenous targets. Analyzing the effect of an siRNA that targets GFP at the single cell level permits a stronger measure of the absolute function of the siRNA by gating to eliminate background levels of GFP intensities. Extending these methods to endogenous proteins, we have shown that well-level results of the knockdown of PTEN results in an increase in phospho-S6 levels, but at the single cell level, the correlation reveals the role of other inputs into the pathway. In a third example, reduction of STAT3 levels by siRNA causes an accumulation of cells in the G1 phase of the cell cycle, but does not induce apoptosis or necrosis when compared to control cells that express the same levels of STAT3. In a final example, the effect of reduced p53 levels on increased adriamycin sensitivity for colon carcinoma cells was demonstrated at the whole-well level using siRNA knockdown and in control and untreated cells at the single cell level.

Conclusion

We find that single cell analysis methods are generally applicable to a wide range of experiments in adherent cells using technology that is becoming increasingly available to most laboratories. It is well-suited to emerging models of signaling dysfunction, such as oncogene addition and oncogenic shock. Single cell cytometry can demonstrate effects on cell function for protein levels that differ by as little as 20%. Biological differences that result from changes in protein level or pathway activation state can be modulated directly by RNAi treatment or extracted from the natural variability intrinsic to cells grown under normal culture conditions.     

RNA interference-mediated survivin gene knockdown induces growth arrest and reduced migration of vascular smooth muscle cells

Survivin (SVV) is a multifunctional protein that has been implicated in the development of neointimal hyperplasia. Nuclear SVV is essential for mitosis, whereas in mitochondria SVV has a cytoprotective function. Here, we investigated the effects of RNA interference (RNAi)-mediated SVV knockdown on cell cycle kinetics, apoptosis, migration, and gene expression in primary cultured vascular smooth muscle cells (VSMCs) from the human saphenous vein. Primary Human VSMCs were obtained from saphenous veins and cultured under standard conditions. SVV knockdown was achieved by either small interfering RNA or lentiviral transduction of short hairpin RNA, reducing SVV gene expression by quantitative PCR (>75%, P < 0.01) without a loss of cell viability. Subcellular fractionation revealed that RNAi treatment effectively targeted the nuclear SVV pool, whereas the larger mitochondrial pool was much less sensitive to transient knockdown. Both p53 and p27 protein levels were notably increased. SVV RNAi treatment significantly blocked VSMC proliferation in response to serum and PDGF-AB, arresting VSMC growth. Cell cycle analysis revealed an increased G(2)/M fraction consistent with a mitotic defect; 4',6-diamidino-2-phenylindole staining confirmed an increased frequency of polyploid and abnormal nuclei. In a transwell assay, SVV knockdown reduced migration to PDGF-AB, and actin-phalloidin staining revealed disorganized actin filaments and polygonal cell shape. However, apoptosis (DNA content and annexin V flow cytometry) was not directly induced by SVV RNAi, and sensitivity to apoptotic agonists (e.g., staurosporine and cytokines) was unchanged. In conclusion, RNAi-mediated SVV knockdown in VSMCs leads to profound cell cycle arrest at G(2)/M and impaired chemotaxis without cytotoxicity. The regulation of mitosis and apoptosis in VSMC involves differentially regulated subcellular pools of SVV. Thus, treatment of VSMC with RNAi targeting SVV might limit the response to vascular injury without destabilizing the vessel wall.