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Murine Embryonic Fibroblasts (murine + embryonic_fibroblast)
Selected AbstractsSequential loss of cell cycle checkpoint control contributes to malignant transformation of murine embryonic fibroblasts induced by 20-methylcholanthreneJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2010Sudeshna Mukherjee Definitive information about the number and nature of discrete steps of tumorigenesis is enigmatic. To understand the multistep nature of carcinogenesis, an in vitro model of 20-Methylcholanthrene-treated primary fibroblast cells CNCI-PM-20, from 20-day old Swiss mouse embryo was used. Visible neoplastic changes with distinct morphological variations along with specific chromosomal aberrations like Robertsonian metacentrics, double and single-minute chromosomes and aneuploidy were observed from Passage-20 onwards. The cell cycle profile showed gradual increase in G2/M population till P-32, followed by evasion of block from P-36 onwards. Gradual increase in expression of C-myc, CyclinD1 and a decrease in expression of P21 was observed from P-20 onwards. CDC25A expression was significantly increased at P-27 and remained more or less constant in subsequent passages. Additionally, an increased P16 and P53 expression were seen at P-20 followed by their significant down-regulation at P-32. An increased level of phosphorylated retinoblastoma (ppRb) was observed from P-27, probably responsible for a compromised G1/S checkpoint. The inactivation of p21 and p16 might be due to their promoter hyper-methylation as suggested through de-methylation experiment by 5-aza-deoxycytidine at P-42. G2/M checkpoint abrogation was marked by gradual increase in expression of CyclinB1 and Cdc20, and a significant increase of Mad2 at P-20. Interestingly, increased expression of phospho-ATM, ATR and phospho-Chk1 were also seen at P-20 followed by their down-regulation at subsequent passages, indicating a perturbation of DNA damage response pathway at early passages. Our findings therefore dramatize the multiple genetic events that can cooperate to promote tumorigenesis. J. Cell. Physiol. 224:49,58, 2010 © 2010 Wiley-Liss, Inc. [source] Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2008G.L. Meng Abstract In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders. Mol. Reprod. Dev. 75: 614,622, 2008. © 2007 Wiley-Liss, Inc. [source] Culturing in vitro produced blastocysts in sequential media promotes ES cell derivationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2006J. Liu Abstract Embryonic stem (ES) cell lines are routinely derived from in vivo produced blastocysts. We investigated the efficiency of ES cells derivation from in vitro produced blastocysts either in monoculture or sequential culture. Zygotes from hybrid F1 B6D2 mice were cultured in vitro to the blastocyst stage in Potassium (K+) simplex optimised medium (KSOM) throughout or in KSOM and switched to COOK blastocyst medium on day 3 (KSOM,CBM). Blastocysts were explanted on a feeder layer of mitomycin C-inactivated murine embryonic fibroblasts (MEF) in TX-WES medium for ES cell derivation. Sequential KSOM,CBM resulted in improved blastocyst formation compared to KSOM monoculture. ES cells were obtained from 32.1% of explanted blastocsyts cultured in KSOM,CBM versus18.4% in KSOM alone. ES cell lines were characterized by morphology, expression of SSEA-1, Oct-4 and alkaline phosphatase activity, and normal karyotype. These results indicate that in vitro culture systems to produce blastocysts can influence the efficiency of ES cell line derivation. Mol. Reprod. Dev. 1017,1021, 2006. © 2006 Wiley-Liss, Inc. [source] Isolation and culture of embryonic stem cells from porcine blastocystsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2003Ming Li Abstract This study was conducted to establish embryonic stem (ES) cell lines from porcine blastocysts. Blastocysts were collected from China miniature pigs at day 7,9 of pregnancy. Embryos were either directly (intact embryos) cultured on mitomysin C-inactivated murine embryonic fibroblasts (MEF) as feeder layers, or were used to isolate the inner cell masses (ICM) by enzyme digestive method and then cultured. It was found that enzyme digestive method could isolate ICMs without any damages of cells in all blastocysts (28). All ICMs attached to the feeder layers. Primary cell colonies were formed in 68% of ICM culture and 28% of intact blastocyst culture. Two ES cell lines derived from ICM passed six subcultures (passages). These cells morphologically resembled mouse ES cells and consistently expressed alkaline phosphatase activity. When the ES cells were cultured in a medium without feeder layer and leukemin inhibitory factor, they differentiated into several types of cells including neuron-like, smooth muscle-like, and epithelium-like cells. Some cells formed embryoid bodies in a suspension culture. These results indicate that porcine ES cell line can be established under the present experimental conditions and these ES cells are pluripotent. Mol. Reprod. Dev. 65: 429,434, 2003. © 2003 Wiley-Liss, Inc. [source] Over-expression of I,B-kinase-, (IKK,/IKKi) induces secretion of inflammatory cytokines in prostate cancer cell linesTHE PROSTATE, Issue 7 2009Benjamin Péant Abstract BACKGROUND Elevated inflammatory cytokine levels in serum have been associated with advanced stage metastasis-related morbidity in prostate cancer. Several studies have shown that IL-6 and IL-8 can accelerate the growth of human prostate cancer cell lines. Previous studies, in murine embryonic fibroblasts, have shown that I,-B kinase-epsilon (IKK,/IKKi)-deficiency results in the reduction of lipopolysaccharide-mediated expression of IL-6. RESULTS In this study, we report that over-expression of IKK, in hormone-sensitive 22Rv1 and LNCaP prostate cancer cells induces the secretion of several inflammatory cytokines including IL-6 and IL-8. Both of these cytokines are secreted by hormone-refractory PC-3 prostate cancer cells and IKK, knock-down in these cells correlates with a strong decrease in IL-6 secretion. Furthermore, we demonstrate that IKK, over-expression does not induce the activation of the IKK, classical targets NF-,B and IRF-3, two transcription factors involved in the regulation of several cytokines. Finally, we observe that high IKK, expression results in its nuclear translocation, a phenomena that is TBK1-independent. CONCLUSIONS This study identifies IKK, as a potential prostate cancer gene that may favor chronic inflammation and create a tumor-supporting microenvironment that promotes prostate cancer progression, particularly by the induction of IL-6 secretion that may act as a positive growth factor in prostate cancer. Prostate 69: 706,718, 2009. © 2009 Wiley-Liss, Inc. [source] | ||||||||||