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Mnt-Max to Myc-Max complex switching regulates cell cycle entry.

Walker W, Zhou ZQ, Ota S, Wynshaw-Boris A, Hurlin PJ - J. Cell Biol. (2005)

Bottom Line: Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes.Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry.These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

View Article: PubMed Central - PubMed

Affiliation: Shriners Hospitals for Children, Portland, OR 97201, USA.

ABSTRACT
The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescued the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

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Mnt overexpression slows cell proliferation and impedes cell cycle entry. (a) Western blot showing expression of Ha-tagged Mnt (anti-Ha set) in pBabeMntHa-infected MEFs (lanes 2 and 4) and endogenous Mnt (anti-Mnt set) in MEFs infected with empty virus (lanes 1 and 3). (b) Proliferation curve conducted for 4 d showing decline in proliferation rate caused by Mnt overexpression in wild-type and Mnt  MEFs. Each value is the average number of cells counted from three different dishes in two separate experiments. (c) Analysis of cell cycle (S-phase) entry in wild-type and Mnt  MEFs overexpressing Mnt. Tritiated thymidine incorporation was measured at the indicated number of hours after serum stimulation of MEFs made quiescent by confluence arrest and serum starvation. Experiments were performed at least twice in triplicate and averages are shown.
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fig4: Mnt overexpression slows cell proliferation and impedes cell cycle entry. (a) Western blot showing expression of Ha-tagged Mnt (anti-Ha set) in pBabeMntHa-infected MEFs (lanes 2 and 4) and endogenous Mnt (anti-Mnt set) in MEFs infected with empty virus (lanes 1 and 3). (b) Proliferation curve conducted for 4 d showing decline in proliferation rate caused by Mnt overexpression in wild-type and Mnt MEFs. Each value is the average number of cells counted from three different dishes in two separate experiments. (c) Analysis of cell cycle (S-phase) entry in wild-type and Mnt MEFs overexpressing Mnt. Tritiated thymidine incorporation was measured at the indicated number of hours after serum stimulation of MEFs made quiescent by confluence arrest and serum starvation. Experiments were performed at least twice in triplicate and averages are shown.

Mentions: The defective cell cycle entry of Mnt MEFs (Hurlin et al., 2003), together with the above findings, suggest that the ratio of Mnt to c-Myc is key to proper cell cycle entry. To better define the relationship between levels of c-Myc and Mnt during cell cycle entry, primary wild-type and Mnt MEFs were infected with Mnt-expressing retrovirus (pBabeMnt-HA), briefly selected to remove noninfected cells, and cell proliferation and cell cycle entry experiments performed. For proliferation assays, cells were plated at 1.5 × 104 cells per 60-mm dish and counted on four consecutive days at 24-h intervals. As shown in Fig. 4 (a and b), Mnt overexpression caused a significant decline in the proliferation rate of wild-type and Mnt−/− MEFs.


Mnt-Max to Myc-Max complex switching regulates cell cycle entry.

Walker W, Zhou ZQ, Ota S, Wynshaw-Boris A, Hurlin PJ - J. Cell Biol. (2005)

Mnt overexpression slows cell proliferation and impedes cell cycle entry. (a) Western blot showing expression of Ha-tagged Mnt (anti-Ha set) in pBabeMntHa-infected MEFs (lanes 2 and 4) and endogenous Mnt (anti-Mnt set) in MEFs infected with empty virus (lanes 1 and 3). (b) Proliferation curve conducted for 4 d showing decline in proliferation rate caused by Mnt overexpression in wild-type and Mnt  MEFs. Each value is the average number of cells counted from three different dishes in two separate experiments. (c) Analysis of cell cycle (S-phase) entry in wild-type and Mnt  MEFs overexpressing Mnt. Tritiated thymidine incorporation was measured at the indicated number of hours after serum stimulation of MEFs made quiescent by confluence arrest and serum starvation. Experiments were performed at least twice in triplicate and averages are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171929&req=5

fig4: Mnt overexpression slows cell proliferation and impedes cell cycle entry. (a) Western blot showing expression of Ha-tagged Mnt (anti-Ha set) in pBabeMntHa-infected MEFs (lanes 2 and 4) and endogenous Mnt (anti-Mnt set) in MEFs infected with empty virus (lanes 1 and 3). (b) Proliferation curve conducted for 4 d showing decline in proliferation rate caused by Mnt overexpression in wild-type and Mnt MEFs. Each value is the average number of cells counted from three different dishes in two separate experiments. (c) Analysis of cell cycle (S-phase) entry in wild-type and Mnt MEFs overexpressing Mnt. Tritiated thymidine incorporation was measured at the indicated number of hours after serum stimulation of MEFs made quiescent by confluence arrest and serum starvation. Experiments were performed at least twice in triplicate and averages are shown.
Mentions: The defective cell cycle entry of Mnt MEFs (Hurlin et al., 2003), together with the above findings, suggest that the ratio of Mnt to c-Myc is key to proper cell cycle entry. To better define the relationship between levels of c-Myc and Mnt during cell cycle entry, primary wild-type and Mnt MEFs were infected with Mnt-expressing retrovirus (pBabeMnt-HA), briefly selected to remove noninfected cells, and cell proliferation and cell cycle entry experiments performed. For proliferation assays, cells were plated at 1.5 × 104 cells per 60-mm dish and counted on four consecutive days at 24-h intervals. As shown in Fig. 4 (a and b), Mnt overexpression caused a significant decline in the proliferation rate of wild-type and Mnt−/− MEFs.

Bottom Line: Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes.Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry.These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

View Article: PubMed Central - PubMed

Affiliation: Shriners Hospitals for Children, Portland, OR 97201, USA.

ABSTRACT
The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescued the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

Show MeSH
Related in: MedlinePlus