Arrest of 3T3 cells in G1 phase in suspension culture

Otsuka, Hidenori; Moskowitz, Merwin

doi:10.1002/jcp.1040870209

Cited by 98 publications

(69 citation statements)

References 10 publications

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“…We previously showed that complementation with this isoform restored the susceptibility of Eker-Cdc6 cells to anchorage loss and destabilized CDC6 (29). Herein, we first confirmed that all of the effects of anchorage deprivation on G 1 (Fig. 3).…”

Section: Expression Of Tsc2 Isoform 4 Restores Susceptibility Of Ekersupporting

confidence: 68%

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Rho-associated Kinase Connects a Cell Cycle-controlling Anchorage Signal to the Mammalian Target of Rapamycin Pathway

Park

Arakawa-Takeuchi

Jinno

et al. 2011

Journal of Biological Chemistry

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When deprived of anchorage to the extracellular matrix, fibroblasts arrest in G 1 phase at least in part due to inactivation of G 1 cyclin-dependent kinases. Despite great effort, how anchorage signals control the G 1 -S transition of fibroblasts remains highly elusive. We recently found that the mammalian target of rapamycin (mTOR) cascade might convey an anchorage signal that regulates S phase entry. Here, we show that Rhoassociated kinase connects this signal to the TSC1/TSC2-RHEB-mTOR pathway. Expression of a constitutively active form of ROCK1 suppressed all of the anchorage deprivation effects suppressible by tsc2 mutation in rat embryonic fibroblasts. TSC2 contains one evolutionarily conserved ROCK target-like sequence, and an alanine substitution for Thr 1203 in this sequence severely impaired the ability of ROCK1 to counteract the anchorage loss-imposed down-regulation of both G 1 cell cycle factors and mTORC1 activity. Moreover, TSC2 Thr 1203 underwent ROCK-dependent phosphorylation in vivo and could be phosphorylated by bacterially expressed active ROCK1 in vitro, providing biochemical evidence for a direct physical interaction between ROCK and TSC2.

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Section: Expression Of Tsc2 Isoform 4 Restores Susceptibility Of Ekersupporting

confidence: 68%

“…The requirement for anchorage is absolute and cannot be overridden by simple growth factor stimulation (1)(2)(3). Anchorage to the ECM is sensed by heterodimeric integrins in the plasma membrane, and a signal is transmitted to various downstream effectors (4,5).…”

mentioning

confidence: 99%

Rho-associated Kinase Connects a Cell Cycle-controlling Anchorage Signal to the Mammalian Target of Rapamycin Pathway

Park

Arakawa-Takeuchi

Jinno

et al. 2011

Journal of Biological Chemistry

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“…Otsuka and Moskowitz (1975) showed that 3T3 cells in S phase could be trypsinized and transferred to a methylcellulose suspension culture without affecting their ability to continue DNA synthesis. Although poorly quantitated, the authors also showed that at least some of these cells gave rise to mitotic figures.…”

Section: Discussionmentioning

confidence: 99%

A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically

Han

Guadagno

Dalton

et al. 1993

The Journal of Cell Biology

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Abstract. We have examined cell cycle control of anchorage-independent growth in nontransformed fibroblasts. In previous studies using Go-synchronized NRK and NIH-3T3 cells, we showed that anchorageindependent growth is regulated by an attachmentdependent transition at G1/S that resembles the START control point in the cell cycle of Saccharomyces cerevisiae. In the studies reported here, we have synchronized NRK and NIH-3T3 fibroblasts immediately after this attachment-dependent transition to determine if other portions of the fibroblast cell cycle are similarly regulated by adhesion. Our results show that S-, G2-, and M-phase progression proceed in the absence of attachment. Thus, we conclude that the adhesion requirement for proliferation of these cells can be explained in terms of the single START-like transition. In related studies, we show that TGF-/~I overrides the attachment-dependent transition in NRK and AKR-2B fibroblasts (lines in which TGF-/$1 induces anchorageindependent growth), but not in NIH-3T3 or Balb/c 3T3 fibroblasts (lines in which TGF-/31 fails to induce anchorage-independent growth). These results show that (a) adhesion and TGF-/31 can have similar effects in stimulating cell cycle progression from G1 to S and (b) the differential effects of TGF-fll on anchorageindependent growth of various fibroblast lines are directly reflected in the differential effects of the growth factor at G1/S. Finally, we have randomly mutagenized NRK fibroblasts to generate mutant lines that have lost their attachment/TGF-~l requirement for G1/S transit while retaining their normal mitogen requirements for proliferation. These clones, which readily proliferate in mitogen-supplemented soft agar, appear non-transformed in monolayer: they are well spread, nonrefractile, and contact inhibited. The existence of this new fibroblast phenotype demonstrates (a) that the growth factor and adhesion/TGF-~/1 requirements for cell cycle progression are genetically separable, (b) that the two major control points in the fibroblast cell cycle (G0/G1 and G1/S) are regulated by distinct extracellular signals, and (c) that the genes regulating anchorage-independent growth need not be involved in regulating contact inhibition, focus formation, or growth factor dependence.

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“…Adhesion deprived NIH3T3 ®broblasts arrest in G1 phase (Otsuka and Moskowitz, 1975) due to down- regulation of many components of the cell cycle machinery , particularly cyclin A (Guadagno et al, 1993;Schulze et al, 1995). Furthermore, growth arrest by contact inhibition is associated with cyclin A down-regulation (Yoshizumi et al, 1995) while other cyclins, such as cyclin D1 continue to be expressed (Meyyappan et al, 1998;Yoshizumi et al, 1995).…”

Section: Rara-plzf Deregulates Cyclin a Expression In Vivomentioning

confidence: 99%

Leukemia translocation protein PLZF inhibits cell growth and expression of cyclin A

et al. 1999

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The PLZF gene was identi®ed by its fusion with the RARa locus in a therapy resistant form of acute promyelocytic leukemia (APL) associated with the t(11;17)(q23;q21) translocation. Here we describe PLZF as a negative regulator of cell cycle progression ultimately leading to growth suppression. PLZF can bind and repress the cyclin A2 promoter while expression of cyclin A2 reverts the growth suppressed phenotype of myeloid cells expressing PLZF. In contrast RARa-PLZF, a fusion protein generated in t(11;17)(q23;q21)-APL activates cyclin A2 transcription and allows expression of cyclin A in anchorage-deprived NIH3T3 cells. Therefore, cyclin A2 is a candidate target gene for PLZF and inhibition of cyclin A expression may contribute to the growth suppressive properties of PLZF. Deregulation of cyclin A2 by RARa-PLZF may represent an oncogenic mechanism of this chimeric protein and contribute to the aggressive clinical phenotype of t(11;17)(q23;q21)-associated APL.

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Arrest of 3T3 cells in G1 phase in suspension culture

Cited by 98 publications

References 10 publications

Rho-associated Kinase Connects a Cell Cycle-controlling Anchorage Signal to the Mammalian Target of Rapamycin Pathway

Rho-associated Kinase Connects a Cell Cycle-controlling Anchorage Signal to the Mammalian Target of Rapamycin Pathway

A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically

Leukemia translocation protein PLZF inhibits cell growth and expression of cyclin A

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