Extended Culture of Mouse Embryo Cells Without Senescence: Inhibition by Serum

Loo, Deryk; Fuquay, J. I.; Rawson, Cathleen; Barnes, David W.

doi:10.1126/science.3494308

Cited by 206 publications

(140 citation statements)

References 18 publications

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“…Mouse embryonic fibroblasts (MEFs) undergo senescence after a limited number of passages in culture, despite their retaining of long telomeres. Elongation of their life span can be achieved by culturing in serum-free medium supplemented with a number of defined growth factors (Loo et al 1987) or by culturing under physiological oxygen conditions (Parrinello et al 2003). Consistent with this, oxidative stress induces cessation of replication in cultured human cells (Packer and Fuehr 1977;Chen et al 1995;Yuan et al 1995), while the replicative potential of human melanocytes and epithelial cells depends largely on the composition of the culture medium used, as well as on the use of feeder layers (Ramirez et al 2001;Bennett and Medrano 2002;Bennett 2003).…”

Section: Stress-induced Senescence In Vitromentioning

confidence: 82%

The essence of senescence: Figure 1.

Kuilman¹,

Michaloglou²,

Mooi³

et al. 2010

Genes Dev.

1,743

1,703

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Almost half a century after the first reports describing the limited replicative potential of primary cells in culture, there is now overwhelming evidence for the existence of “cellular senescence” in vivo. It is being recognized as a critical feature of mammalian cells to suppress tumorigenesis, acting alongside cell death programs. Here, we review the various features of cellular senescence and discuss their contribution to tumor suppression. Additionally, we highlight the power and limitations of the biomarkers currently used to identify senescent cells in vitro and in vivo.

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Section: Stress-induced Senescence In Vitromentioning

confidence: 82%

The essence of senescence: Figure 1.

Kuilman¹,

Michaloglou²,

Mooi³

et al. 2010

Genes Dev.

1,743

1,703

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show abstract

“…In fact, serum is believed to contribute to a phenomenon known as "culture shock," in which mammalian cells are exposed to an unrelenting onslaught of mitogenic signals that may lead to DNA damage, checkpoint activation, and senescence (for review, see Sherr and DePinho 2000). To overcome the pitfalls associated with culture shock, we adapted cell culture conditions similar to those described previously (Barnes and Sato 1980;Loo et al 1987), where serum was replaced with epidermal growth factor (EGF), insulin, high density lipoprotein (HDL), and transferrin (see Materials and Methods for more details).…”

Section: Mefs Grown In Serum-free Conditions Are Immortalmentioning

confidence: 99%

“…Similarly, MEFs grown in the absence of serum, but with a specific cocktail of supplements (including EGF, PDGF, insulin, transferrin, HDL, and fibronectin), do not undergo senescence. These cells proliferate indefinitely without any of the significant karyotypic alterations seen in established mouse cell lines (Loo et al 1987).…”

mentioning

confidence: 99%

Activated oncogenes promote and cooperate with chromosomal instability for neoplastic transformation

Woo¹,

Poon²

2004

Genes Dev.

112

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Most cancer cells are aneuploid. The chromosomal instability hypothesis contends that aneuploidy is the catalyst for transformation, whereas the gene mutation hypothesis asserts that cancer is driven by mutations to proto-oncogenes and tumor-suppressor genes, with the aneuploidy a side effect of tumorigenesis. Because genotoxic stress induced by "culture shock" can obscure the transforming potential of exogenous genes, we cultured wild-type and p53 −/− mouse embryo fibroblasts in a more physiological (serum-free) environment. Under these conditions, the cells were immortal and, more importantly, chromosomally stable. Expression of oncogenic H-RasV12 did not induce senescence, but sensitized these cells to p53-dependent apoptosis. In addition, H-RasV12 induced chromosomal instability, as well as accumulation and phosphorylation of p53. Significantly, whereas cells grown under standard conditions could be transformed by coexpression of H-RasV12 and E1A, the chromosomally stable cells were refractory to transformation, as measured by anchorage-independent growth and tumor formation in nude mice. These oncogenes required a third genetic alteration that abolished the p53 pathway to create a permissive environment that promotes rapid chromosomal instability and transformation. Oncogene-induced chromosomal instability and transformation was attenuated by antioxidants. These data indicate that chromosomal instability could be a catalyst for oncogenic transformation, and bring together aspects of the chromosomal instability hypothesis and the gene mutation hypothesis for tumorigenesis.[Keywords: Chromosomal instability; oncogenic Ras; p53; transformation] Supplemental material is available at http://www.genesdev.org.

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“…Indeed, under more gentle and more defined culture conditions, primary mouse cells can be convinced to proliferate for extended periods of time in vitro (9). Tissue culture stress signals induce expression of a number of anti-proliferative genes, including p16 INK4A and p19 ARF (10,11).…”

mentioning

confidence: 99%

Reversal of Senescence in Mouse Fibroblasts through Lentiviral Suppression of p53

Dirac¹,

Bernards

2003

Journal of Biological Chemistry

228

163

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Senescence is generally defined as an irreversible state of G 1 cell cycle arrest in which cells are refractory to growth factor stimulation. In mouse embryo fibroblasts (MEFs), induction of senescence requires the presence of p19 ARF and p53, as genetic ablation of either of these genes allows escape from senescence and leads to immortalization. We have developed a lentiviral vector that directs the synthesis of a p53-specific short hairpin transcript, which mediates stable suppression of p53 expression through RNA interference. We show that suppression of p53 expression in senescent MEFs leads to rapid cell cycle re-entry, is associated with loss of expression of senescence-associated genes, and leads to immortalization. These data indicate that senescence in MEFs is reversible and demonstrate that both initiation and maintenance of senescence is p53-dependent.

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Extended Culture of Mouse Embryo Cells Without Senescence: Inhibition by Serum

Cited by 206 publications

References 18 publications

The essence of senescence: Figure 1.

The essence of senescence: Figure 1.

Activated oncogenes promote and cooperate with chromosomal instability for neoplastic transformation

Reversal of Senescence in Mouse Fibroblasts through Lentiviral Suppression of p53

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