Murine erythroleukemia cell differentiation: relationship of globin gene expression and of prolongation of G1 to inducer effects during G1/early S.

Gambari, Roberto; Marks, Paul A.; Rifkind, Richard A.

doi:10.1073/pnas.76.9.4511

Cited by 55 publications

(32 citation statements)

References 24 publications

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“…Commitment has been thought to be limited to a specific phase of the cell cycle (4,6,18). Herbimycin-treated TSA8 cells can pass through one S phase but are arrested at the Gi phase and at the G2/M boundary (Fig.…”

Section: Discussionmentioning

confidence: 99%

Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver cells and murine erythroleukemia (TSA8) cells without proliferation.

et al. 1988

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Erythropoietin (epo) appears to play a significant role in influencing the proliferation and differentiation of erythroid progenitor (CFU-E) cells. To determine the mechanism of action of epo, the effect of drugs on the in vitro colony formation of CFU-E cells induced from a novel murine erythroleukemia cell line, TSA8, was examined. While cytosine arabinoside inhibited colony formation and terminal differentiation of the CFU-E cells responding to epo, herbimycin, which is a drug that inhibits src-related phosphorylation, inhibited colony formation only. The same effect of herbimycin was observed with normal CFU-E cells from mouse fetal liver cells. These results suggest that epo induces two signals, one for proliferation and the other for differentiation, and that the two signals are not linked in erythroid progenitor cells.

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Section: Discussionmentioning

confidence: 99%

Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver cells and murine erythroleukemia (TSA8) cells without proliferation.

et al. 1988

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“…Hexamethylenebisacetamide (HMBA; ref. 19) was added to 4 mM and the mixture was incubated for 6 hr to arrest cells in G1. Flow cytometry of the HMBA-arrested cells, carried out with the Becton Dickinson Immunocytometry system, showed that -90%o of cells were in G1 phase.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of the p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase.

Pan

Amin²,

Gibbs³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The human single-stranded-DNA-binding protein (HSSB, also called RP-A) is a trimeric complex (p70, p34, and p14) required for multiple functions in DNA transactions. We report here that the p34 subunit of HSSB was hyperphosphorylated by kinase activities present in G1 extract (obtained from HeLa cells in G1 phase) preincubated with human cyclin A. This hyperphosphorylated HSSB product included at least four species of p34 that migrated more slowly through denaturing polyacrylamide gels than the hypophosphorylated form. Fractionation ofcyclin A-activated G, extract identified two kinases involved in the hyperphosphorylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p35O protein kinase (DNA-PK). Kinetic analysis revealed that in cyclin A-activated G1 extract, p34 was first phosphorylated by cdk-cyclin A prior to the action of DNA-PK. Addition of p2ldPl, a specific inhibitor of cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation of HSSB p34 in G, extract preincubated with cyclin A. This suggests a requirement of the cdk-cyclin A activity for the phosphorylation ofp34 by DNA-PK in G, extract. We report that HSSB p34 was hyperphosphorylated in G1 extract preincubated with human cyclin A and that this was due to the combined actions ofthe cdk-cyclin A complex and the p350 DNA-dependent protein kinase (DNA-PK). MATERIALS AND METHODSProtein Reagents. HSSB, human cyclin A, and p2lciPl were purified as described (12,17,18), and cdc2-cyclin B from sea star and protein phosphatase 2Ac were purchased from Upstate Biotechnology (Lake Placid, NY).Preparation of G, Extract. Twenty liters of HeLa cells, grown to 5-6 x 105 cells per ml at 370C in Joklik's medium with 5% bovine calf serum, were treated with nocodazole (40 ng/ml) for 12 hr to arrest cells at the G2/M boundary. The cells were centrifuged at 1300 x g for 5 min at 220C, washed free of drug by gentle resuspension in 4 liters of phosphatebuffered saline, and centrifuged again. Pelleted cells were gently suspended in 16 liters of Joklik's medium with 10%o bovine calf serum and then allowed to recover for 2 hr at 370C. Hexamethylenebisacetamide (HMBA; ref. 19) was added to 4 mM and the mixture was incubated for 6 hr to arrest cells in G1. Flow cytometry of the HMBA-arrested cells, carried out with the Becton Dickinson Immunocytometry system, showed that -90%o of cells were in G1 phase. Cytosolic extract of G1 cells was prepared as described (12).Immunoblot Assays. Monoclonal antibodies (mAbs) against HSSB p34 (12) were used for immunoblot analysis (20).Kinase Asays. For the phosphorylation of HSSB with G1 extract, reaction mixtures (10 y4) containing 40 mM creatine phosphate (pH 7.7), creatine kinase at 25 ,ug/ml, 7 mM MgCl2, 0.5 mM dithiothreitol, 4 mM ATP, G1 extracts, and cyclin A were incubated at 370C as indicated.For the phosphorylation of HSSB p34 by purified kinases, 8343The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance...

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“…An early manifestation of termination of cell division in MELC appears to be a transient prolongation of the GI phase of the cell cycle, which can be detected after cell transit through one complete S phase in the presence of inducer (8)(9)(10). After up to 4-5 terminal cell divisions, induced MELC are arrested in a GI-like phase of the cell cycle.…”

mentioning

confidence: 99%

Protein p53 and inducer-mediated erythroleukemia cell commitment to terminal cell division.

Shen¹,

Real²,

DeLeo³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Inducer-mediated murine erythroleukemia cell (MELC) differentiation provides a model for examining factors determining terminal cell differentiation. The nuclear protein, p53, has been implicated as a potential determinant of cell cycle progression and cell differentiation. In this study p53 content and synthesis, during inducer-mediated MELC differentiation, has been examined with monoclonal antibodies to p53. A decrease in p53 synthesis and content was demonstrated during induced differentiation. As determined by cell cycle fractionation, the decrease in p53 is manifest at all stages of the cell cycle. Hemin, which induces globin mRNA accumulation but not terminal cell division, fails to decrease p53 content. A MELC variant resistant to inducer-mediated commitment to terminal cell division also fails to decrease p53 levels in response to inducers. These experiments suggest that p53 is implicated in MELC cell proliferation and that an induced decrease in p53 may be responsible for GI phase prolongation and terminal GI arrest.The murine erythroleukemia cell (MELC), a virus-transformed erythroid cell precursor approximating the erythroid colonyforming unit (designated CFUe) stage in the erythropoietic lineage (1), provides a model for study of regulatory mechanisms responsible for coordinate unlinked gene expression (a-and 13-globin mRNA accumulation) and initiation of terminal cell divisions (termed commitment) during cell differentiation (2, 3). MELC can be induced by hexamethylene bisacetamide (HMBA), dimethylsulfoxide (Me2SO), or other chemical agents to initiate a program of differentiation that appears similar to that of normal erythroid precursors (1, 4). Of particular advantage is the availability of different inducers capable of initiating distinct developmental patterns (5, 6) and variant cell lines resistant to inducer-mediated commitment to terminal cell division but competent to express other features of erythroid differentiation (7).An early manifestation of termination of cell division in MELC appears to be a transient prolongation of the GI phase of the cell cycle, which can be detected after cell transit through one complete S phase in the presence of inducer (8-10). After up to 4-5 terminal cell divisions, induced MELC are arrested in a GI-like phase of the cell cycle. Evidence has accumulated suggesting that synthesis of a labile protein during G1 may control entry of cells into S phase (11). A nuclear protein (p53 protein), initially recognized by its elevated levels in transformed cells (12) and by its capacity to bind to simian virus 40 (SV40) tumor antigen (13,14), has been identified as possibly important in determining normal cell cycle progression from G1 to S (15, 16). It also has been suggested that p53 protein levels may be regulated in relation to the differentiation status of a cell lineage (17,18). Based upon a number of observations, it appears that cellular p53 content is determined by both transcriptional and post-transcriptional mechanisms (17)(18)(19)(20). The present...

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Murine erythroleukemia cell differentiation: relationship of globin gene expression and of prolongation of G1 to inducer effects during G1/early S.

Cited by 55 publications

References 24 publications

Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver cells and murine erythroleukemia (TSA8) cells without proliferation.

Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver cells and murine erythroleukemia (TSA8) cells without proliferation.

Phosphorylation of the p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase.

Protein p53 and inducer-mediated erythroleukemia cell commitment to terminal cell division.

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