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CCAAT/enhancer binding protein alpha uses distinct domains to prolong pituitary cells in the growth 1 and DNA synthesis phases of the cell cycle.

Liu W, Enwright JF, Hyun W, Day RN, Schaufele F - BMC Cell Biol. (2002)

Bottom Line: C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells.Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Metabolic Research Unit, Diabetes Research Center and Department of Medicine, University of California, San Francisco, CA 94143-0540, USA. liu_weiqun@hotmail.com

ABSTRACT

Background: A number of transcription factors coordinate differentiation by simultaneously regulating gene expression and cell proliferation. CCAAT/enhancer binding protein alpha (C/EBPalpha) is a basic/leucine zipper transcription factor that integrates transcription with proliferation to regulate the differentiation of tissues involved in energy balance. In the pituitary, C/EBPalpha regulates the transcription of a key metabolic regulator, growth hormone.

Results: We examined the consequences of C/EBPalpha expression on proliferation of the transformed, mouse GHFT1-5 pituitary progenitor cell line. In contrast to mature pituitary cells, GHFT1-5 cells do not contain C/EBPalpha. Ectopic expression of C/EBPalpha in the progenitor cells resulted in prolongation of both growth 1 (G1) and the DNA synthesis (S) phases of the cell cycle. Transcription activation domain 1 and 2 of C/EBPalpha were required for prolongation of G1, but not of S. Some transcriptionally inactive derivatives of C/EBPalpha remained competent for G1 and S phase prolongation. C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.

Conclusion: We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells. G1 and S phase prolongation did not require that C/EBPalpha remained transcriptionally active or retained the ability to dimerize via the leucine zipper. G1, but not S, arrest required a domain overlapping with C/EBPalpha transcription activation functions 1 and 2. Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

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GHFT1-5 cells were grown to subconfluence and treated for 20 hours with DMSO (white bars), 100 ng/ml nocodazole (gray bars) or 0.5 mM mimosine (speckled gray bars), both in DMSO. The amount of DNA within each cell was measured by flow cytometric quantification of the orange fluorescence emitted from propidium iodide-stained DNA. The proportion of GHFT1-5 cells containing a 2n complement of DNA (G1), a 4n complement of DNA (G2/M), and an intermediate amount of DNA (S) was determined for each treatment and presented as the mean +/- standard deviation from nine independent experiments.
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Figure 3: GHFT1-5 cells were grown to subconfluence and treated for 20 hours with DMSO (white bars), 100 ng/ml nocodazole (gray bars) or 0.5 mM mimosine (speckled gray bars), both in DMSO. The amount of DNA within each cell was measured by flow cytometric quantification of the orange fluorescence emitted from propidium iodide-stained DNA. The proportion of GHFT1-5 cells containing a 2n complement of DNA (G1), a 4n complement of DNA (G2/M), and an intermediate amount of DNA (S) was determined for each treatment and presented as the mean +/- standard deviation from nine independent experiments.

Mentions: Transcriptionally active C/EBPα-GFP and transcriptionally inactive GFP-C/EBPα were compared for their effects on proliferation of GHFT1-5 cells. We first determined the distribution of untransfected GHFT1-5 cells in each phase of the cell cycle. GHFT1-5 cells were grown to subconfluence, collected and the DNA within the cells was stained with propidium iodide. The DNA content within each cell was quantified by flow cytometry as the amount of orange fluorescence from the propidium iodide-stained DNA (see Materials and Methods for details). The fluorescence intensity measured from each cell falls into one of three populations: cells centered around the lowest level of orange fluorescence, cells centered around the highest level of orange fluorescence which is twice that of the lowest, and cells with intermediate levels of orange fluorescence. This corresponds, respectively, to cells containing a 2n DNA complement prior to duplication of the genome (in G1 phase), cells containing a 4n DNA complement following genome duplication (in growth phase 2 or in mitosis, G2/M), and cells containing a partially replicated genome intermediate between 2n and 4n (in S phase). As averaged from nine independent experiments, the proportion of growing GHFT1-5 cells in G1, S and G2/M corresponded to 42%, 38% and 20%, respectively (Fig 3, white bars).


CCAAT/enhancer binding protein alpha uses distinct domains to prolong pituitary cells in the growth 1 and DNA synthesis phases of the cell cycle.

Liu W, Enwright JF, Hyun W, Day RN, Schaufele F - BMC Cell Biol. (2002)

GHFT1-5 cells were grown to subconfluence and treated for 20 hours with DMSO (white bars), 100 ng/ml nocodazole (gray bars) or 0.5 mM mimosine (speckled gray bars), both in DMSO. The amount of DNA within each cell was measured by flow cytometric quantification of the orange fluorescence emitted from propidium iodide-stained DNA. The proportion of GHFT1-5 cells containing a 2n complement of DNA (G1), a 4n complement of DNA (G2/M), and an intermediate amount of DNA (S) was determined for each treatment and presented as the mean +/- standard deviation from nine independent experiments.
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Related In: Results  -  Collection

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Figure 3: GHFT1-5 cells were grown to subconfluence and treated for 20 hours with DMSO (white bars), 100 ng/ml nocodazole (gray bars) or 0.5 mM mimosine (speckled gray bars), both in DMSO. The amount of DNA within each cell was measured by flow cytometric quantification of the orange fluorescence emitted from propidium iodide-stained DNA. The proportion of GHFT1-5 cells containing a 2n complement of DNA (G1), a 4n complement of DNA (G2/M), and an intermediate amount of DNA (S) was determined for each treatment and presented as the mean +/- standard deviation from nine independent experiments.
Mentions: Transcriptionally active C/EBPα-GFP and transcriptionally inactive GFP-C/EBPα were compared for their effects on proliferation of GHFT1-5 cells. We first determined the distribution of untransfected GHFT1-5 cells in each phase of the cell cycle. GHFT1-5 cells were grown to subconfluence, collected and the DNA within the cells was stained with propidium iodide. The DNA content within each cell was quantified by flow cytometry as the amount of orange fluorescence from the propidium iodide-stained DNA (see Materials and Methods for details). The fluorescence intensity measured from each cell falls into one of three populations: cells centered around the lowest level of orange fluorescence, cells centered around the highest level of orange fluorescence which is twice that of the lowest, and cells with intermediate levels of orange fluorescence. This corresponds, respectively, to cells containing a 2n DNA complement prior to duplication of the genome (in G1 phase), cells containing a 4n DNA complement following genome duplication (in growth phase 2 or in mitosis, G2/M), and cells containing a partially replicated genome intermediate between 2n and 4n (in S phase). As averaged from nine independent experiments, the proportion of growing GHFT1-5 cells in G1, S and G2/M corresponded to 42%, 38% and 20%, respectively (Fig 3, white bars).

Bottom Line: C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells.Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Metabolic Research Unit, Diabetes Research Center and Department of Medicine, University of California, San Francisco, CA 94143-0540, USA. liu_weiqun@hotmail.com

ABSTRACT

Background: A number of transcription factors coordinate differentiation by simultaneously regulating gene expression and cell proliferation. CCAAT/enhancer binding protein alpha (C/EBPalpha) is a basic/leucine zipper transcription factor that integrates transcription with proliferation to regulate the differentiation of tissues involved in energy balance. In the pituitary, C/EBPalpha regulates the transcription of a key metabolic regulator, growth hormone.

Results: We examined the consequences of C/EBPalpha expression on proliferation of the transformed, mouse GHFT1-5 pituitary progenitor cell line. In contrast to mature pituitary cells, GHFT1-5 cells do not contain C/EBPalpha. Ectopic expression of C/EBPalpha in the progenitor cells resulted in prolongation of both growth 1 (G1) and the DNA synthesis (S) phases of the cell cycle. Transcription activation domain 1 and 2 of C/EBPalpha were required for prolongation of G1, but not of S. Some transcriptionally inactive derivatives of C/EBPalpha remained competent for G1 and S phase prolongation. C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.

Conclusion: We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells. G1 and S phase prolongation did not require that C/EBPalpha remained transcriptionally active or retained the ability to dimerize via the leucine zipper. G1, but not S, arrest required a domain overlapping with C/EBPalpha transcription activation functions 1 and 2. Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

Show MeSH
Related in: MedlinePlus