Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor Nocodazole

Zieve, Gary W.; Turnbull, Deborah; Mullins, J. Michael; McIntosh, J. Richard

doi:10.1016/0014-4827(80)90279-7

Cited by 382 publications

(219 citation statements)

References 17 publications

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“…The half-life for the return of Dab2 to the dephosphorylated state was approximately 1.5 h, as shown in the quantification of the data presented in Figure 5b. This time frame corresponded well with the time it took for the cells to completely proceed through mitosis and exit mitosis phase, according to our own flow cytometry studies (data not shown) and the previous work of others on the exit of HeLa cells from nocodazole-induced blockade of mitosis (Zieve et al, 1980). The dephosphorylation of Dab2 following exit from mitosis phase was completely blocked by incubation of the cells with juglone, an inhibitor of Pin1 peptidylprolyl isomerase activity (Figure 6a).…”

Section: Dephosphorylation Of Dab2 Is Dependent On Pin1 and Pp1/pp2asupporting

confidence: 88%

Cell cycle-dependent phosphorylation of Disabled-2 by cdc2

et al. 2003

Oncogene

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Disabled-2 (Dab2; also known as p96 and DOC-2) is a signal transduction protein that has been implicated in the control of cell growth. Dab2 is known to be a phosphoprotein, but little is known about the kinases that phosphorylate Dab2. We have found that Dab2 phosphorylation is markedly increased during the mitosis phase of the cell cycle. This phosphorylation is blocked by roscovitine, a selective inhibitor of cyclin-dependent kinases. Dab2 robustly coimmunoprecipitates from cells with the cyclin-dependent kinase cdc2, and purified cdc2 can phosphorylate purified Dab2 fusion proteins in vitro on multiple sites. Cellular phosphorylation of Dab2 by cdc2 promotes the association of Dab2 with Pin1, a peptidylprolyl isomerase that regulates the rate of Dab2 dephosphorylation. These findings reveal that Dab2 is differentially phosphorylated during the cell cycle by cdc2 and provide a potential feedback mechanism by which Dab2 inhibition of cell growth and proliferation may be regulated.

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Section: Dephosphorylation Of Dab2 Is Dependent On Pin1 and Pp1/pp2asupporting

confidence: 88%

Cell cycle-dependent phosphorylation of Disabled-2 by cdc2

et al. 2003

Oncogene

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“…To clarify whether or not G1 arrest occurred after p53 induction in SCLC cells, we added nocodazol to p53-induced cells. Nocodazol is one of rapidly-reversible inhibitors of microtubule polymerization which can block the cell cycle at G2/M phase (Zieve et al, 1980;Cross et al, 1995;Ookawa et al, 1997), and thus prevents the cells recycling back from the G2/M phase to the G1 phase. Cells exposed to the induced state for 24 h were treated with nocodazol for additional 12, 24 and 48 h and were analyzed by flow cytometry (Figure 3).…”

Section: Resultsmentioning

confidence: 99%

Phenotypic alterations of small cell lung carcinoma induced by different levels of wild-type p53 expression

et al. 1998

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p53 induces both growth arrest and apoptosis in cancer cells.To clarify whether the level of p53 expression determines the response of small cell lung carcinoma (SCLC) cells, we assessed the effect of various p53 levels on a p53-null SCLC cell line, N417, using a tetracycline (Tc)-regulated inducible p53 expression system. Apoptosis was induced in SCLC cells with high p53 expression. Although low levels of p53 induced G1 arrest accompanied by p21 expression, cells with G1 arrest seemed to undergo apoptosis after further cultivation. Expression of exogenous p21 induced G1 arrest but not apoptosis in SCLC cells, suggesting that p53-mediated G1 arrest was induced through p21 expression. Moreover, high level of p53 expression down-regulated Bcl-2 expression in SCLC cells, while Bax was consistently expressed irrespective to the level of p53 expression. These results suggest that p53-mediated apoptosis and G1 arrest depend on level of p53 expression in SCLC cells and that the relative dominancy of Bax to Bcl-2 is involved in the induction of apoptosis by high level of p53 expression.

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“…As shown, anti-NuMA-2 sera did not show IIF staining of interphase cells, and the antigen recognized by these autoantibodies was only consistently detected in mitotic cells. Considering that the majority of cells in asynchronous culture are in interphase, HeLa S3 cells were synchronized in mitosis (35) and used as immunoblot substrate. Control IIF with anti-NuMA-2 serum Sr showed that the vast majority of cells in such culture (85-90%) were at metaphasetelophase, with good staining of the mitotic apparatus.…”

Section: Resultsmentioning

confidence: 99%

“…For preparation of whole cell lysate, subconfluent HeLa S3 cells were harvested and immediately solubilized in Laemmli sample buffer (LSB) (34). Substrate enriched in mitotic cells was obtained through a prometaphase-arrest protocol (35). Briefly, subconfluent HeLa S3 cells grown on large Petri dishes were incubated with 10 mM thymidine for 16 hours, washed in PBS, and incubated with regular medium for 5 hours.…”

Section: Methodsmentioning

confidence: 99%

Two major autoantigen—Antibody systems of the mitotic spindle apparatus

Andrade

Chan

Peebles

et al. 1996

Arthritis & Rheumatism

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Objective. To characterize human autoantigenantibody systems related to the mitotic poles and spindles.Methods. Thirty-seven human sera with autoantibodies staining mitotic poles and spindles in indirect immunofluorescence (IIF) studies were further characterized by immunofluorescence on mitotic cells and by immunoblotting and immunoprecipitation. Clinical diagnoses meeting the American College of Rheumatology criteria were based on chart review and interview with the corresponding physicians.ResuUs. Two autoantibody systems reactive with mitotic poles and spindles were defined. Type 1 nuclear mitotic apparatus (NuMA-1) antibodies were identified in the serum of 30 patients. Interphase cells showed a fine, speckled, nuclear staining, while mitotic cells had bright staining of the rim of the centrosomes and light staining of the spindles proximal to the centrosomes. In telophase, the staining shifted from the centrosomes to the reforming nuclei. On immunoblotting, antiNuMA-1 sera reacted with a 210-kd protein. The reactivity of these sera was identified (with the aid of reference antibodies) as the previously described NuMA antigen-antibody system. Clinical information was available for only 17 of the 30 patients with antiNuMA-1; of these, 17 (53%) had clinical and lip biopsy findings that met the criteria for Sjiigren's syndrome.Publication 7668-MEM from The Scripps Research Institute.

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Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor Nocodazole

Cited by 382 publications

References 17 publications

Cell cycle-dependent phosphorylation of Disabled-2 by cdc2

Cell cycle-dependent phosphorylation of Disabled-2 by cdc2

Phenotypic alterations of small cell lung carcinoma induced by different levels of wild-type p53 expression

Two major autoantigen—Antibody systems of the mitotic spindle apparatus

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