A Cross-talk between oncogenic Ras and tumor suppressor PTEN through FAK Tyr861 phosphorylation in NIH/3T3 mouse embryonic fibroblasts

ABSTRACT The cell cycle inhibitor p15 INK4b is frequently inactivated by homozygous deletion together with p16 INK4a and p19 ARF in some types of tumors. Although the tumor suppressor capability of p15 INK4b is still questioned, it has been found to be specifically inactivated by hypermethylation in hematopoietic malignancies in the absence of p16 INK4a alterations. Here we show that, in vitro, p15 INK4b is a strong inhibitor of cellular transformation by Ras. Surprisingly, p15 INK4b is induced in cultured cells by oncogenic Ras to an extent similar to that of p16 INK4a , and their expression is associated with premature G1 arrest and senescence. Ras-dependent induction of these two INK4 genes is mediated mainly by the Raf-Mek-Erk pathway. Studies with activated and dominant negative forms of Ras effectors indicate that the Raf-Mek-Erk pathway is essential for induction of both the p15 INK4b and p16 INK4a promoters, although other Ras effector pathways can collaborate, giving rise to a stronger response. Our results indicate that p15 INK4b , by itself, is able to stop cell transformation by Ras and other oncogenes such as Rgr (a new oncogene member of the Ral-GDS family, whose action is mediated through Ras). In fact, embryonic fibroblasts isolated from p15 INK4b knockout mice are susceptible to transformation by the Ras or Rgr oncogene whereas wild-type embryonic fibroblasts are not. Similarly, p15 INK4b -deficient mouse embryo fibroblasts are more sensitive than wild-type cells to transformation by a combination of the Rgr and E1A oncogenes. The cell cycle inhibitor p15 INK4b is therefore involved, at least in some cell types, in the tumor suppressor activity triggered after inappropriate oncogenic Ras activation in the cell.

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Expression of Caveolin-1 Induces Premature Cellular Senescence in Primary Cultures of Murine Fibroblasts

Molecular Biology of the Cell ◽

10.1091/mbc.01-11-0529 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2502-2517 ◽

Cited By ~ 146

Author(s):

Daniela Volonte ◽

Kun Zhang ◽

Michael P. Lisanti ◽

Ferruccio Galbiati

Keyword(s):

Hydrogen Peroxide ◽

Cellular Senescence ◽

Premature Senescence ◽

3T3 Cells ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Caveolin 1 ◽

Senescent Phenotype ◽

Nih 3T3

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a “transformation suppressor” protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G0/G1 phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in β-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when caveolin-1 levels are restored. Taken together, these results clearly indicate a central role for caveolin-1 in promoting cellular senescence and they suggest the hypothesis that premature senescence may represent a tumor suppressor function mediated by caveolin-1 in vivo.

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Chemical composition of the essential oil from basil (Ocimum basilicumLinn.) and itsin vitrocytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts

Natural Product Research ◽

10.1080/14786419.2010.545357 ◽

2012 ◽

Vol 26 (12) ◽

pp. 1112-1118 ◽

Cited By ~ 43

Author(s):

Poonkodi Kathirvel ◽

Subban Ravi

Keyword(s):

Chemical Composition ◽

Essential Oil ◽

Cell Lines ◽

Cancer Cell ◽

Human Cancer ◽

Human Cancer Cell ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Human Cancer Cell Lines ◽

Nih 3T3

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Promoter activity of earthworm metallothionein in mouse embryonic fibroblasts

Molecular Biology Reports ◽

10.1007/s11033-019-05082-1 ◽

2019 ◽

Vol 46 (6) ◽

pp. 6371-6379

Author(s):

Victoria Drechsel ◽

Birgit Fiechtner ◽

Martina Höckner

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Promoter Activity ◽

Gene Activation ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Transfection Experiment ◽

Activation Mechanisms ◽

Nih 3T3 ◽

Transcription Factor 1

Abstract The regulation of metallothionein (MT) gene expression as important part of the detoxification machinery is only scarcely known in invertebrates. In vertebrates, MT gene activation is mediated by the metal-transcription factor 1 (MTF-1) binding to metal response elements (MREs). In invertebrates, the mechanisms of MT gene activation seems to be more diverse. In some invertebrate species, MTF-1 orthologues as well as their ability to activate MT genes via MREs have been uncovered. Although earthworm MTs have been well studied, a MTF-1 orthologue has not yet been described and MT gene activation mechanisms are largely unknown. Analyses of the earthworm wMT2 promoter by reporter gene assays have been performed. We could show that the wMT2 promoter was active in mouse embryonic fibroblasts (NIH/3T3) as well as in mouse MTF-1−/−cells (DKO7). The presence of mouse MTF-1 (mMTF1) led to a significant increase in reporter gene activity. We observed that cadmium as well as zinc had an effect on promoter activity. In the presence of zinc, promoter activity doubled in NIH cells, however, we did not observe a significant effect in the DKO7 cell line. Cadmium decreased promoter activity in DKO7 cells, but this effect could be reversed by providing mMTF1 in a co-transfection experiment. We suggest that MT gene expression in the earthworm is not entirely dependent on a MRE binding protein. Interestingly, the shortest promoter fragment including MRE1 showed the highest promoter activity under control conditions.

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SIRT6 regulates Ras-related protein R-Ras2 by lysine defatty-acylation

eLife ◽

10.7554/elife.25158 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 30

Author(s):

Xiaoyu Zhang ◽

Nicole A Spiegelman ◽

Ornella D Nelson ◽

Hui Jing ◽

Hening Lin

Keyword(s):

Tumor Suppressor ◽

Related Protein ◽

Tumor Suppressor Function ◽

Phosphatidylinositol 3 Kinase ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

Unknown Mechanism ◽

Fatty Acylation ◽

Established Tumor ◽

Ras Family

The Ras family of GTPases are important in cell signaling and frequently mutated in human tumors. Understanding their regulation is thus important for studying biology and human diseases. Here, we report that a novel posttranslational mechanism, reversible lysine fatty acylation, regulates R-Ras2, a member of the Ras family. SIRT6, a sirtuin with established tumor suppressor function, regulates the lysine fatty acylation of R-Ras2. In mouse embryonic fibroblasts (MEFs), Sirt6 knockout (KO) increased R-Ras2 lysine fatty acylation. Lysine fatty acylation promotes the plasma membrane localization of R-Ras2 and its interaction with phosphatidylinositol 3-kinase PI3K, leading to activated Akt and increased cell proliferation. Our study establishes lysine fatty acylation as a previously unknown mechanism that regulates the Ras family of GTPases and provides an important mechanism by which SIRT6 functions as a tumor suppressor.

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Prep1 and Meis1 competition for Pbx1 binding regulates protein stability and tumorigenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1321200111 ◽

2014 ◽

Vol 111 (10) ◽

pp. E896-E905 ◽

Cited By ~ 29

Author(s):

Leila Dardaei ◽

Elena Longobardi ◽

Francesco Blasi

Keyword(s):

Tumor Suppressor ◽

Integration Site ◽

Viral Integration ◽

Transcriptional Level ◽

Cell Leukemia ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

B Cell Leukemia ◽

Growth Promoting ◽

Different Levels

Pbx-regulating protein-1 (Prep1) is a tumor suppressor, whereas myeloid ecotropic viral integration site-1 (Meis1) is an oncogene. We show that, to perform these activities in mouse embryonic fibroblasts, both proteins competitively heterodimerize with pre–B-cell leukemia homeobox-1 (Pbx1). Meis1 alone transforms Prep1-deficient fibroblasts, whereas Prep1 overexpression inhibits Meis1 tumorigenicity. Pbx1 can, therefore, alternatively act as an oncogene or tumor suppressor. Prep1 posttranslationally controls the level of Meis1, decreasing its stability by sequestering Pbx1. The different levels of Meis1 and the presence of Prep1 are followed at the transcriptional level by the induction of specific transcriptional signatures. The decrease of Meis1 prevents Meis1 interaction with Ddx3x and Ddx5, which are essential for Meis1 tumorigenesis, and modifies the growth-promoting DNA binding landscape of Meis1 to the growth-controlling landscape of Prep1. Hence, the key feature of Prep1 tumor-inhibiting activity is the control of Meis1 stability.

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