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Overexpression of RasG12V in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We have performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21Waf1/Cip1 rescues from RasG12V-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented RasG12V-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from RasG12V-induced senescence by prevention of RasG12V-induced upregulation of p21Waf1/Cip1. Our results establish an important role for the cell cycle inhibitor p21Waf1/Cip1 in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour suppressor. [ABSTRACT FROM AUTHOR]

Overexpression of RasG12V in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We have performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21Waf1/Cip1 rescues from RasG12V-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented RasG12V-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from RasG12V-induced senescence by prevention of RasG12V-induced upregulation of p21Waf1/Cip1. Our results establish an important role for the cell cycle inhibitor p21Waf1/Cip1 in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour suppressor. [ABSTRACT FROM AUTHOR]

Abstract Background Upon cellular entry retroviruses must avoid innate restriction factors produced by the host cell. For human immunodeficiency virus (HIV) human restriction factors, APOBEC3 (apolipoprotein-B-mRNA-editing-enzyme), p21 and tetherin are well characterised. Results To identify intrinsic resistance factors to HIV-1 replication we screened 19,121 human genes and identified 114 factors with significant inhibition of infection. Those with a known function are involved in a broad spectrum of cellular processes including receptor signalling, vesicle trafficking, transcription, apoptosis, cross-nuclear membrane transport, meiosis, DNA damage repair, ubiquitination and RNA processing. We focused on the PAF1 complex which has been previously implicated in gene transcription, cell cycle control and mRNA surveillance. Knockdown of all members of the PAF1 family of proteins enhanced HIV-1 reverse transcription and integration of provirus. Over-expression of PAF1 in host cells renders them refractory to HIV-1. Simian Immunodeficiency Viruses and HIV-2 are also restricted in PAF1 expressing cells. PAF1 is expressed in primary monocytes, macrophages and T-lymphocytes and we demonstrate strong activity in MonoMac1, a monocyte cell line. Conclusions We propose that the PAF1c establishes an anti-viral state to prevent infection by incoming retroviruses. This previously unrecognised mechanism of restriction could have implications for invasion of cells by any pathogen.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Exploration of the human melanoma cell-cycle pathway can lead to identification of new therapeutic targets. By gene set enrichment analysis, we identified the cell-cycle pathway and its member polo-like kinase 1 (Plk-1) to be significantly overexpressed in primary melanomas and in melanoma metastases. In vitro expression of Plk-1 was peaked at the G2/M phase of the cell cycle. Plk-1 knockdown/inhibition led to induction of apoptosis, which was caspase-3/8-dependent and p53-independent, and involved BID and Bcl-2 proteins. Comparative genomic hybridization/single-nucleotide polymorphism arrays showed no genetic alteration in the Plk-1 locus. Previous suggestions and significant enrichment of the mitogen-activated protein kinase (MAPK) signaling pathway pointed to potential regulation of Plk-1 by MAPK signaling. Inhibition of this pathway resulted in decreased Plk-1 expression as a consequence of G1 cell-cycle arrest rather than direct regulation of Plk-1. Inhibition of MAPK and Plk-1 had an additive effect on reduced cell viability. This study shows that in human melanoma, Plk-1 expression is dynamically regulated during the cell cycle, knockdown of Plk-1 leads to apoptotic cell death, and that a combination of Plk-1 and MAPK inhibition has an additive effect on melanoma cell viability. We conclude that combined inhibition of Plk-1 and MAPK could be a potentially attractive strategy in melanoma therapy.

In a genome-wide siRNA analysis of p16(INK4a) (p16) modulators, we identify the Hedgehog (Hh) pathway component SUFU and formally demonstrate that Hh signaling promotes mitogenesis by suppression of p16. A fragment of the Hh-responsive GLI2 transcription factor directly binds and inhibits the p16 promoter and senescence is associated with the loss of nuclear GLI2. Hh components partially reside in the primary cilium (PC), and the small fraction of cells in mass culture that elaborate a PC have the lowest expression of p16. Suppression of p16 is effected by both PC-dependent and -independent routes, and ablation of p16 renders cells insensitive to an Hh inhibitor and increases PC formation. These results directly link a well-established developmental mitogenic pathway with a key tumor suppressor and contribute to the molecular understanding of replicative senescence, Hh-mediated oncogenesis, and potentially the role of p16 in aging., (Copyright © 2010 Elsevier Inc. All rights reserved.)

To investigate involvement of DNA mismatch repair in the response to short-wave ultraviolet (UVC) light, we compared UVC-induced mutant frequencies and mutational spectra at the Hprt gene between wild type and mismatch-repair-deficient mouse embryonic stem (ES) cells. Whereas mismatch repair gene status did not significantly affect survival of these cells after UVC irradiation, UVC induced substantially more mutations in ES cells that lack the MutSalpha mismatch-recognizing heterodimer than in wild type ES cells. The global UVC-induced mutational spectra at Hprt and the distribution of most spectral mutational hotspots were found to be similar in mismatch-repair-deficient and wild type cells. However, at one predominant spectral hot spot for mutagenesis in wild type cells, the UVC-induced mutation frequency was not affected by the mismatch repair status. Together these data reveal a major role of mismatch repair in controlling mutagenesis induced by UVC light and may suggest the sequence context-dependent direct mismatch repair of misincorporations opposite UVC-induced pyrimidine dimers.

We have developed a simple procedure that enables the efficient selection of cells that are deficient for DNA mismatch repair (MMR). This selection procedure was used to investigate the frequency of fortuitous MMR-deficient cells in a mouse embryonic stem cell line, heterozygous for the MMR gene Msh2. We found a surprisingly high frequency (3 x 10(-4)) of Msh2-deficient cells. The wild type Msh2 allele was almost invariably lost by loss of heterozygosity. Single treatments with the genotoxic agents ethylnitrosourea, UVC light and mitomycin C resulted in a further increase of the number of Msh2-/- cells in the heterozygous cell line. This increase was not only due to induced loss of the wild type allele but also to a selective growth advantage of preexisting Msh2-/- cells to ethylnitrosourea and UVC. Mitomycin C, in contrast to ethylnitrosourea and UVC, uniquely induced loss of heterozygosity at Msh2. These mechanistically different ways of loss of the wild type Msh2 allele reflect the different repair pathways processing these damages. Heterozygous germ line defects in one of the MMR genes underlie the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. Based on the results described here we hypothesize that mutagen-induced loss of MMR in the intestine of these patients contributes to the tissue specificity of carcinogenesis in HNPCC patients.