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Homozygous deletion of glycogen synthase kinase 3beta bypasses senescence allowing Ras transformation of primary murine fibroblasts.

ABSTRACT: In primary mammalian cells, expression of oncogenes such as activated Ras induces premature senescence rather than transformation. We show that homozygous deletion of glycogen synthase kinase (GSK) 3beta (GSK3beta-/-) bypasses senescence induced by mutant Ras(V12) allowing primary mouse embryo fibroblasts (MEFs) as well as immortalized MEFs to exhibit a transformed phenotype in vitro and in vivo. Both catalytic activity and Axin-binding of GSK3beta are required to optimally suppress Ras transformation. The expression of Ras(V12) in GSK3beta-/-, but not in GSK3beta+/+ MEFs results in translocation of beta-catenin to the nucleus with concomitant up-regulation of cyclin D1. siRNA-mediated knockdown of beta-catenin decreases both cyclin D1 expression and anchorage-independent growth of transformed cells indicating a causal role for beta-catenin. Thus Ras(V12) and the lack of GSK3beta act in concert to activate the beta-catenin pathway, which may underlie the bypass of senescence and tumorigenic transformation by Ras.

SUBMITTER: Liu S

PROVIDER: S-EPMC2278194 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Homozygous deletion of glycogen synthase kinase 3beta bypasses senescence allowing Ras transformation of primary murine fibroblasts.

Liu Shuying S Fang Xianjun X Hall Hassan H Yu Shuangxing S Smith Debra D Lu Zhimin Z Fang Dexing D Liu Jinsong J Stephens L Clifton LC Woodgett James R JR Mills Gordon B GB

Proceedings of the National Academy of Sciences of the United States of America 20080326 13

In primary mammalian cells, expression of oncogenes such as activated Ras induces premature senescence rather than transformation. We show that homozygous deletion of glycogen synthase kinase (GSK) 3beta (GSK3beta-/-) bypasses senescence induced by mutant Ras(V12) allowing primary mouse embryo fibroblasts (MEFs) as well as immortalized MEFs to exhibit a transformed phenotype in vitro and in vivo. Both catalytic activity and Axin-binding of GSK3beta are required to optimally suppress Ras transfor ...[more]

PMID: 18367674

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Project description:The MYC and RAS oncogenes are sufficient for transformation of normal rodent cells. This cooperativity is at least in part based on suppression of RAS-induced cellular senescence by MYC and block of MYC-induced apoptosis by RAS - thereby canceling out two main barriers against tumor development. However, it remains unclear whether MYC and RAS cooperate in this way in human cells, where MYC and RAS are not sufficient for transformation. To address this question, we established a combined Tet-inducible H-RASV12 and hydroxytamoxifen-inducible MycER system in normal human BJ fibroblasts. We show here that activation of RAS alone induced senescence while activation of MYC alone or together with RAS triggered DNA damage, induction of p53 and massive apoptosis, suggesting that RAS cannot rescue MYC-induced apoptosis in this system. Although coexpression with MYC reduced certain RAS-induced senescence markers (histone H3 lysine 9 trimethylation and senescence-associated ?-GAL activity), the induction of the senescence marker p16INK4A was further enhanced and the culture ceased to proliferate within a few days, revealing that MYC could not fully suppress RAS-induced senescence. Furthermore, depletion of p53, which enhanced proliferation and rescued the cells from RAS-induced senescence, did not abrogate MYC-induced apoptosis. We conclude that MYC and RAS are unable to cooperate in overcoming senescence and apoptosis in normal human fibroblasts even after depletion of p53, indicating that additional oncogenic events are required to abrogate these fail-safe mechanisms and pave the way for cellular transformation. These findings have implications for our understanding of the transformation process in human cells. Abbreviations and acronyms: CDK: Cyclin-dependent kinase; DDR: DNA damage response; DOX: Doxycycline; EdU: 5-ethynyl-2'-deoxyuridine; FACS: Fluorescence Activated Cell Sorting; MycER: MYC-estrogen receptor; OHT: 4-hydroxytamoxifen; OIS: Oncogene-induced senescence; PP2A: Protein phosphatase 2A; ROS: Reactive oxygen species; SA-?-GAL: Senescence-associated ?-galactosidase; SAHF: Senescence-associated heterochromatin foci; shRNA: Short hairpin RNA; YFP: Yellow fluorescent protein.

Dataset Information

Homozygous deletion of glycogen synthase kinase 3beta bypasses senescence allowing Ras transformation of primary murine fibroblasts.

Publications

Homozygous deletion of glycogen synthase kinase 3beta bypasses senescence allowing Ras transformation of primary murine fibroblasts.

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