How to become immortal: let MEFs count the ways

Odell, Adam F.; Askham, Jon M.; Whibley, Catherine; Hollstein, Monica

doi:10.18632/aging.100129

Cited by 37 publications

(48 citation statements)

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“…2F). It is well known that wild-type p53 is one of the master regulators in cellular senescence (Odell et al 2010). The decreased expression of wild-type p53 in PCBP4-deficient low-passage primary MEFs might lead to partial senescence bypass in MEFs, as observed in Figure 2, C and D. Consistently, we also found that the level of p53 protein was lower in the PCBP4 −/− mouse thymus (Fig.…”

Section: Pcbp4-deficient Mice Are Prone To Lung Adenocarcinoma and Lysupporting

confidence: 87%

Mice deficient in poly(C)-binding protein 4 are susceptible to spontaneous tumors through increased expression of ZFP871 that targets p53 for degradation

et al. 2016

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Poly(C)-binding protein 4 (PCBP4), also called MCG10 and a target of p53, plays a role in the cell cycle and is implicated in lung tumor suppression. Here, we found that PCBP4-deficient mice are prone to lung adenocarcinoma, lymphoma, and kidney tumor and that PCBP4-deficient mouse embryo fibroblasts (MEFs) exhibit enhanced cell proliferation but decreased cellular senescence. We also found that p53 expression is markedly reduced in PCBP4-deficient MEFs and mouse tissues, suggesting that PCBP4 in turn regulates p53 expression. To determine how PCBP4 regulates p53 expression, PCBP4 targets were identified by RNA immunoprecipitation followed by RNA sequencing (RNA-seq). We found that the transcript encoding ZFP871 (zinc finger protein 871; also called ZNF709 in humans) interacts with and is regulated by PCBP4 via mRNA stability. Additionally, we found that ZFP871 physically interacts with p53 and MDM2 proteins. Consistently, ectopic expression of ZFP871 decreases-whereas knockdown of ZFP871 increases-p53 protein stability through a proteasome-dependent degradation pathway. Moreover, loss of ZFP871 reverses the reduction of p53 expression by lack of PCBP4, and thus increased expression of ZFP871 is responsible for decreased expression of p53 in the PCBP4-deficient MEFs and mouse tissues. Interestingly, we found that, like PCBP4, ZFP871 is also regulated by DNA damage and p53. Finally, we showed that knockdown of ZFP871 markedly enhances p53 expression, leading to growth suppression and apoptosis in a p53-dependent manner. Thus, the p53-PCBP4-ZFP871 axis represents a novel feedback loop in the p53 pathway. Together, we hypothesize that PCBP4 is a potential tissue-specific tumor suppressor and that ZFP871 is part of MDM2 and possibly other ubiquitin E3 ligases that target p53 for degradation.

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Section: Pcbp4-deficient Mice Are Prone To Lung Adenocarcinoma and Lysupporting

confidence: 87%

Mice deficient in poly(C)-binding protein 4 are susceptible to spontaneous tumors through increased expression of ZFP871 that targets p53 for degradation

et al. 2016

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“…Historically, human fibroblast lines established from patients biopsies have been used in a number of studies to elucidate the pathogenesis of several diseases (Villegas and McPhaul 2005). Ever since the establishment of HeLa cells by George Gey in 1951 (Jones et al 1971), scientists established several other fibroblast lines for different use, such as the fibroblasts NIH3T3 from Swiss mouse derived embryos, in which fibroblast were transformed with the antigen T from SV40 virus, turning them into a growing stable cell line (Todaro and Green, 2010), the cell line HEK293T, used in the production of viral vectors (Acrani et al 2010), or primary cultures of fibroblasts derived from mouse embryos (MEF) used as feeder for embryonic stem cells culture (Odell et al 2010.;vom Brocke, et al 2006).…”

Section: Introductionmentioning

confidence: 99%

Fibroblast sources: Where can we get them?

et al. 2014

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Fibroblasts are cells widely used in cell culture, both for transient primary cell culture or permanent as transformed cell lines. Lately, fibroblasts become cell sources for use in disease modeling after cell reprogramming because it is easily accessible in the body. Fibroblasts in patients will maintain all genetic background during reprogramming into induced pluripotent stem cells. In spite of their large use, fibroblasts are obtained after an invasive procedure, a superficial punch skin biopsy, collected under patient's local anesthesia. Taking into consideration the minimum patient's discomfort during and after the biopsy procedure, as well as the aesthetics aspect, it is essential to reflect on the best site of the body for the biopsy procedure combined with the success of getting robust fibroblast cultures in the lab. For this purpose, we compared the efficiency of four biopsy sites of the body (skin from eyelid, back of the ear, abdominal cesarean scar and groin). Cell proliferation assays and viability after cryopreservation were measured. Our results revealed that scar tissue provided fibroblasts with higher proliferative rates. Also, fibroblasts from scar tissues presented a higher viability after the thawing process.

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“…Previous studies have detected TP53 mutations in up to 30% of mutagen‐treated cultures or 5 to 20% of spontaneously immortalised cultures 10, 11, 12, 13. The remaining cultures are TP53 ‐WT and likely harbour alterations in other genes associated with senescence‐bypass 14, 15…”

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confidence: 99%

Nutlin‐3a selects for cells harbouring TP53 mutations

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Hollstein

Arlt

et al. 2016

Intl Journal of Cancer

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TP53 mutations occur in half of all human tumours. Mutagen‐induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock‐in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen‐treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2–5 months) and much effort is expended maintaining TP53‐WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin‐3a, an MDM2 inhibitor that leads to stabilisation and activation of wild‐type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin‐3a to examine the effect on cell growth and p53 activation. Nutlin‐3a induced the p53 pathway in TP53‐WT HUFs and inhibited cell growth, whereas most TP53‐mutated HUFs were resistant to Nutlin‐3a. We then assessed whether Nutlin‐3a treatment could discriminate between TP53‐WT and TP53‐mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin‐3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin‐3a‐resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin‐3a‐sensitive clones were TP53‐WT. These data suggest that including a Nutlin‐3a counter‐screen significantly improves the specificity and efficiency of the HIMA, whereby TP53‐mutated clones are selected prior to sequencing and TP53‐WT clones can be discarded.

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How to become immortal: let MEFs count the ways

Cited by 37 publications

References 38 publications

Mice deficient in poly(C)-binding protein 4 are susceptible to spontaneous tumors through increased expression of ZFP871 that targets p53 for degradation

Mice deficient in poly(C)-binding protein 4 are susceptible to spontaneous tumors through increased expression of ZFP871 that targets p53 for degradation

Fibroblast sources: Where can we get them?

Nutlin‐3a selects for cells harbouring TP53 mutations

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