Cell Cycle-Related Differences in Susceptibility of NIH/3T3 Cells to Ribonucleases

ABSTRACT Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.

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Cell Cycle Progression Regulates Biogenesis and Cellular Localization of Lipid Droplets

Molecular and Cellular Biology ◽

10.1128/mcb.00374-18 ◽

2019 ◽

Vol 39 (9) ◽

Cited By ~ 4

Author(s):

André L. S. Cruz ◽

Nina Carrossini ◽

Leonardo K. Teixeira ◽

Luis F. Ribeiro-Pinto ◽

Patricia T. Bozza ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Lipid Accumulation ◽

Lipid Droplets ◽

Cell Cycle Progression ◽

Cellular Transformation ◽

3T3 Cells ◽

Cycle Progression ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACTIntracellular lipid accumulation has been associated with a poor prognosis in cancer. We have previously reported the involvement of lipid droplets in cell proliferation in colon cancer cells, suggesting a role for these organelles in cancer development. In this study, we evaluate the role of lipid droplets in cell cycle regulation and cellular transformation. Cell cycle synchronization of NIH 3T3 cells revealed increased numbers and dispersed distribution of lipid droplets specifically during S phase. Also, the transformed cell lineage NIH 3T3-H-rasV12showed an accumulation of both lipid droplets and PLIN2 protein above the levels in NIH 3T3 cells.PLIN2gene overexpression, however, was not able to induce NIH 3T3 cell transformation, disproving the hypothesis thatPLIN2is an oncogene. Furthermore, positive PLIN2 staining was strongly associated with highly proliferative Ki-67-positive areas in human colon adenocarcinoma tissue samples. Taken together, these results indicate that cell cycle progression is associated with tight regulation of lipid droplets, a process that is altered in transformed cells, suggesting the existence of a mechanism that connects cell cycle progression and cell proliferation with lipid accumulation.

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Ras Signals to the Cell Cycle Machinery via Multiple Pathways To Induce Anchorage-Independent Growth

Molecular and Cellular Biology ◽

10.1128/mcb.18.5.2586 ◽

1998 ◽

Vol 18 (5) ◽

pp. 2586-2595 ◽

Cited By ~ 47

Author(s):

Jaw-Ji Yang ◽

Jong-Sun Kang ◽

Robert S. Krauss

Keyword(s):

Cell Cycle ◽

Colony Formation ◽

Cyclin A ◽

Soft Agar ◽

Specific Cell ◽

3T3 Cells ◽

Anchorage Independent Growth ◽

Nih 3T3 ◽

Cell Cycle Machinery ◽

Nih 3T3 Cells

ABSTRACT Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.

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Unusual proliferation arrest and transcriptional control properties of a newly discovered E2F family member, E2F-6

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.16.9190 ◽

1998 ◽

Vol 95 (16) ◽

pp. 9190-9195 ◽

Cited By ~ 110

Author(s):

Stefan Gaubatz ◽

Jason G. Wood ◽

David M. Livingston

Keyword(s):

Cell Cycle ◽

Transcriptional Control ◽

Cell Cycle Progression ◽

Specific Effect ◽

3T3 Cells ◽

Cycle Progression ◽

E2f Transcription Factors ◽

Regulation Of Cell Cycle ◽

Nih 3T3 ◽

Nih 3T3 Cells

E2F transcription factors play an important role in the regulation of cell cycle progression. We report here the cloning and characterization of an additional member of this family, E2F-6. E2F-6 lacks pocket protein binding and transactivation domains, and it is a potent transcriptional repressor that contains a modular repression domain at its carboxyl terminus. Overproduction of E2F-6 had no specific effect on cell cycle progression in asynchronously growing Saos2 and NIH 3T3 cells, but it inhibited entry into S phase of NIH 3T3 cells stimulated to exit G0. Taken together, these data suggest that E2F-6 can regulate a subset of E2F-dependent genes whose products are required for entry into the cell cycle but not for normal cell cycle progression.

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pp39mos is associated with p34cdc2 kinase in c-mosxe-transformed NIH 3T3 cells

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3583-3589.1992 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3583-3589

Author(s):

R Zhou ◽

I Daar ◽

D K Ferris ◽

G White ◽

R S Paules ◽

...

Keyword(s):

Cell Cycle ◽

Histone H1 ◽

Molar Ratio ◽

3T3 Cells ◽

Peptide Antibody ◽

Tight Association ◽

Nih 3T3 ◽

Nih 3T3 Cells

We investigated the possible interactions between pp39mos and p34cdc2 kinase in NIH 3T3 cells transformed by c-mosxe. pp39mos is coprecipitated with p34cdc2 when using either anti-PSTAIR antibody or p13suc1-Sepharose beads. Likewise, p34cdc2 is coprecipitated with pp39mos when using anti-mos antibody. However, pp39mos was not present in histone H1 kinase-active p34cdc2 complexes precipitated with anti-p34cdc2 C-terminal peptide antibody even during metaphase of the cell cycle. The molar ratio of p34 to pp39mos in the p13suc1 complex is approximately 2:1. Consistent with the tight association between pp39mos and tubulin, tubulin was also present in equivalent amounts with pp39mos and p34 in the p13suc1 complex. This pp39mos-p34cdc2-tubulin complex may be important in transformation by the mos oncogene.

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pp39mos is associated with p34cdc2 kinase in c-mosxe-transformed NIH 3T3 cells.

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3583 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3583-3589 ◽

Cited By ~ 10

Author(s):

R Zhou ◽

I Daar ◽

D K Ferris ◽

G White ◽

R S Paules ◽

...

Keyword(s):

Cell Cycle ◽

Histone H1 ◽

Molar Ratio ◽

3T3 Cells ◽

Peptide Antibody ◽

Tight Association ◽

Nih 3T3 ◽

Nih 3T3 Cells

We investigated the possible interactions between pp39mos and p34cdc2 kinase in NIH 3T3 cells transformed by c-mosxe. pp39mos is coprecipitated with p34cdc2 when using either anti-PSTAIR antibody or p13suc1-Sepharose beads. Likewise, p34cdc2 is coprecipitated with pp39mos when using anti-mos antibody. However, pp39mos was not present in histone H1 kinase-active p34cdc2 complexes precipitated with anti-p34cdc2 C-terminal peptide antibody even during metaphase of the cell cycle. The molar ratio of p34 to pp39mos in the p13suc1 complex is approximately 2:1. Consistent with the tight association between pp39mos and tubulin, tubulin was also present in equivalent amounts with pp39mos and p34 in the p13suc1 complex. This pp39mos-p34cdc2-tubulin complex may be important in transformation by the mos oncogene.

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Fibroblast growth factor-1 stimulation of quiescent NIH 3T3 cells increases G/T mismatch-binding protein expression

Biochemical Journal ◽

10.1042/bj3190009 ◽

1996 ◽

Vol 319 (1) ◽

pp. 9-12 ◽

Cited By ~ 6

Author(s):

Patrick J. DONOHUE ◽

Sheau-Line Y. FENG ◽

Gregory F ALBERTS ◽

Yan GUO ◽

Kimberly A PEIFLEY ◽

...

Keyword(s):

Cell Cycle ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Binding Protein ◽

Calf Serum ◽

Mrna Levels ◽

Fibroblast Growth ◽

3T3 Cells ◽

Nih 3T3 ◽

Nih 3T3 Cells

Polypeptide growth factors promote cell-cycle progression in part by the transcriptional activation of a diverse group of specific genes. We have used an mRNA differential-display approach to identify several fibroblast growth factor (FGF)-1 (acidic FGF)-inducible genes in NIH 3T3 cells. Here we report that one of these genes, called FGF-regulated (FR)-3, is predicted to encode G/T mismatch-binding protein (GTBP), a component of the mammalian DNA mismatch correction system. The murine GTBP gene is transiently expressed after FGF-1 or calf serum treatment, with maximal mRNA levels detected at 12 and 18 h post-stimulation. FGF-1-stimulated NIH 3T3 cells also express an increased amount of GTBP as determined by immunoblot analysis. These results indicate that elevated levels of GTBP may be required during the DNA synthesis phase of the cell cycle for efficient G/T mismatch recognition and repair.

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