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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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Deletion of Mnt rescues proliferation arrest caused by loss of c-Myc. (a) PCR genotyping of MEFs of the indicated genotypes after infection with empty (E) adenovirus or Cre (C) recombinase-expressing adenovirus. Note the PCR primers used to detect deleted Mnt alleles do not discriminate from wild-type and recombined Mnt alleles. (b) Western blot examining Mnt and c-Myc expression after AdCre infection of the indicated genotypes. (c and d) MEF proliferation curves after deletion of Mnt, c-Myc, and Mnt plus c-Myc in primary MEFs (c) and immortal MEFs (d). Values shown in c and d are the average of cell counts obtained from triplicate plates and are representative of results obtained from two independent experiments. (e) Cell cycle profiles generated from FAC sorting of propidium iodide stained cells 48 h after AdCre infection of the indicated cell lines. % of cells in S-phase (S) is shown. Arrows highlight sub G1 (growth phase 1) fractions consistent with apoptosis.
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fig5: Deletion of Mnt rescues proliferation arrest caused by loss of c-Myc. (a) PCR genotyping of MEFs of the indicated genotypes after infection with empty (E) adenovirus or Cre (C) recombinase-expressing adenovirus. Note the PCR primers used to detect deleted Mnt alleles do not discriminate from wild-type and recombined Mnt alleles. (b) Western blot examining Mnt and c-Myc expression after AdCre infection of the indicated genotypes. (c and d) MEF proliferation curves after deletion of Mnt, c-Myc, and Mnt plus c-Myc in primary MEFs (c) and immortal MEFs (d). Values shown in c and d are the average of cell counts obtained from triplicate plates and are representative of results obtained from two independent experiments. (e) Cell cycle profiles generated from FAC sorting of propidium iodide stained cells 48 h after AdCre infection of the indicated cell lines. % of cells in S-phase (S) is shown. Arrows highlight sub G1 (growth phase 1) fractions consistent with apoptosis.

Mentions: To better define the relationship between Mnt and c-Myc in cell proliferation and cell cycle entry, we examined the consequences of acute, simultaneous deletion of both Mnt and c-Myc in primary MEFs. Crosses between Mntflox/flox (Hurlin et al., 2003) and c-MycfloxN/floxN mice (Trumpp et al., 2001) were performed to generate mice containing homozygous floxed alleles for both Mnt and c-Myc (Mnt/c-MycdCKO). MEFs were then isolated from the latter mice and from Mntflox/flox mice and c-Mycflox/flox mice. MEFs at passage 1 were infected with adenovirus expressing Cre recombinase and GFP. GFP expression indicated that >95% of cells were infected and PCR genotyping and Western blot analysis confirmed that a very high percentage of floxed alleles were successfully targeted (Fig. 5, a and b, not depicted). Proliferation assays performed 48 h after infection cells confirmed previous results (de Alboran et al., 2001; Trumpp et al., 2001) showing that deletion of c-Myc leads to rapid cessation of primary MEF proliferation (Fig. 5 c). In contrast, primary MEFs in which c-Myc and Mnt were simultaneously ablated continued to proliferate (Fig. 5 c). Very similar results were achieved after AdCre infection of immortal (i) Mntflox/flox, ic-Mycflox/flox, and iMnt-c-MycdCKO MEF cell lines (Fig. 5 d). The latter cell lines were developed by continuously passaging primary cell populations until they escaped senescence (Todaro and Green, 1963).


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)

Deletion of Mnt rescues proliferation arrest caused by loss of c-Myc. (a) PCR genotyping of MEFs of the indicated genotypes after infection with empty (E) adenovirus or Cre (C) recombinase-expressing adenovirus. Note the PCR primers used to detect deleted Mnt alleles do not discriminate from wild-type and recombined Mnt alleles. (b) Western blot examining Mnt and c-Myc expression after AdCre infection of the indicated genotypes. (c and d) MEF proliferation curves after deletion of Mnt, c-Myc, and Mnt plus c-Myc in primary MEFs (c) and immortal MEFs (d). Values shown in c and d are the average of cell counts obtained from triplicate plates and are representative of results obtained from two independent experiments. (e) Cell cycle profiles generated from FAC sorting of propidium iodide stained cells 48 h after AdCre infection of the indicated cell lines. % of cells in S-phase (S) is shown. Arrows highlight sub G1 (growth phase 1) fractions consistent with apoptosis.
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Related In: Results  -  Collection

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fig5: Deletion of Mnt rescues proliferation arrest caused by loss of c-Myc. (a) PCR genotyping of MEFs of the indicated genotypes after infection with empty (E) adenovirus or Cre (C) recombinase-expressing adenovirus. Note the PCR primers used to detect deleted Mnt alleles do not discriminate from wild-type and recombined Mnt alleles. (b) Western blot examining Mnt and c-Myc expression after AdCre infection of the indicated genotypes. (c and d) MEF proliferation curves after deletion of Mnt, c-Myc, and Mnt plus c-Myc in primary MEFs (c) and immortal MEFs (d). Values shown in c and d are the average of cell counts obtained from triplicate plates and are representative of results obtained from two independent experiments. (e) Cell cycle profiles generated from FAC sorting of propidium iodide stained cells 48 h after AdCre infection of the indicated cell lines. % of cells in S-phase (S) is shown. Arrows highlight sub G1 (growth phase 1) fractions consistent with apoptosis.
Mentions: To better define the relationship between Mnt and c-Myc in cell proliferation and cell cycle entry, we examined the consequences of acute, simultaneous deletion of both Mnt and c-Myc in primary MEFs. Crosses between Mntflox/flox (Hurlin et al., 2003) and c-MycfloxN/floxN mice (Trumpp et al., 2001) were performed to generate mice containing homozygous floxed alleles for both Mnt and c-Myc (Mnt/c-MycdCKO). MEFs were then isolated from the latter mice and from Mntflox/flox mice and c-Mycflox/flox mice. MEFs at passage 1 were infected with adenovirus expressing Cre recombinase and GFP. GFP expression indicated that >95% of cells were infected and PCR genotyping and Western blot analysis confirmed that a very high percentage of floxed alleles were successfully targeted (Fig. 5, a and b, not depicted). Proliferation assays performed 48 h after infection cells confirmed previous results (de Alboran et al., 2001; Trumpp et al., 2001) showing that deletion of c-Myc leads to rapid cessation of primary MEF proliferation (Fig. 5 c). In contrast, primary MEFs in which c-Myc and Mnt were simultaneously ablated continued to proliferate (Fig. 5 c). Very similar results were achieved after AdCre infection of immortal (i) Mntflox/flox, ic-Mycflox/flox, and iMnt-c-MycdCKO MEF cell lines (Fig. 5 d). The latter cell lines were developed by continuously passaging primary cell populations until they escaped senescence (Todaro and Green, 1963).

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