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Overexpression of E2F5/p130, but not E2F5 alone, can inhibit E2F-induced cell cycle entry in transgenic mice.

Chen Q, Liang D, Overbeek PA - Mol. Vis. (2008)

Bottom Line: However, coexpression of E2F5/p130 with E2F1 or E2F3a in lens fiber cells decreased the number of BrdU positive fiber cells induced by the E2F1 or E2F3a alone.Overexpression of E2F5/p130 in post-mitotic lens fiber cells does not affect their normal differentiation program, but can inhibit inappropriate cell cycle reentry induced by the activator E2Fs.Since E2F5 alone cannot interfere with these E2F activities, we conclude that p130 is a key player in the inhibitory process.

View Article: PubMed Central - PubMed

Affiliation: College of Optometry, University of Houston, Houston, TX 77204-2020, USA.

ABSTRACT

Purpose: The retinoblastoma (Rb) gene family member p130 binds preferentially to the E2F5 transcription factor and forms a repressive E2F5/p130 complex that inhibits cell cycle progression and tumor growth. It is unclear whether E2F5, either alone or in combination with p130, can interfere with the transcriptional activity of other E2F family members, such as E2F1 and E2F3a, in vivo. In this study, we used transgenic mice to test whether overexpression of E2F5 with/without p130 would be sufficient to inhibit E2F1 or E2F3a induced cell cycle reentry.

Methods: Transgenic mice were generated by microinjection of constructs containing different E2F cDNAs (E2F1, E2F3a, and E2F5) or the p130 cDNA linked to the mouse alphaA-crystallin promoter. The E2F5 single and E2F5/p130 double transgenic mice were cross-mated with E2F1 or E2F3a transgenic mice. The resulting double or triple transgenic mouse embryos were characterized by histology, in situ hybridization, immunohistochemistry, and BrdU incorporation assays.

Results: Overexpression of E2F5 alone was not sufficient to inhibit E2F1 or E2F3a induced cell cycle reentry in lens fiber cells. Transgenic mice coexpressing E2F5 and p130 in lens fiber cells did not show lens defects. However, coexpression of E2F5/p130 with E2F1 or E2F3a in lens fiber cells decreased the number of BrdU positive fiber cells induced by the E2F1 or E2F3a alone. Therefore, overexpression of E2F5/p130, but not E2F5 alone, can inhibit activator E2F-mediated cell proliferation in vivo, confirming that p130 plays a critical role in the repressive activity of E2F5/p130 complex.

Conclusions: Overexpression of E2F5/p130 in post-mitotic lens fiber cells does not affect their normal differentiation program, but can inhibit inappropriate cell cycle reentry induced by the activator E2Fs. Since E2F5 alone cannot interfere with these E2F activities, we conclude that p130 is a key player in the inhibitory process.

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E2F3a/E2F5/p130 triple transgenic lens histology and BrdU incorporation assays. A-C: At E15.5, when compared with a non-transgenic FVB lens (A), the triple transgenic lens showed a modest phenotype with some displaced fiber cell nuclei(C) in contrast to the many fiber cell nuclei seen in the E2F3a single transgenic lens (B). Abbreviations: le, lens epithelium; lf, lens fiber. D-F: The number of BrdU positive fiber cells (arrow head) in the triple transgenic lens (F) decreased by about 50% when compared to the E2F3a single transgenic lens (E). Scale bars=500 μm.
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f7: E2F3a/E2F5/p130 triple transgenic lens histology and BrdU incorporation assays. A-C: At E15.5, when compared with a non-transgenic FVB lens (A), the triple transgenic lens showed a modest phenotype with some displaced fiber cell nuclei(C) in contrast to the many fiber cell nuclei seen in the E2F3a single transgenic lens (B). Abbreviations: le, lens epithelium; lf, lens fiber. D-F: The number of BrdU positive fiber cells (arrow head) in the triple transgenic lens (F) decreased by about 50% when compared to the E2F3a single transgenic lens (E). Scale bars=500 μm.

Mentions: Hypophosphorylated p130 forms a repressor complex with the E2F4, which can block E2F-responsive promoter sites, and can inhibit the G1/S transition of the cell cycle [18-20]. We hypothesized that the E2F5/p130 complex could also function as a cell cycle repressor in vivo. To examine whether coexpression of E2F5 and p130 can inhibit cell proliferation induced by E2F3a, E2F3a transgenic mice (family OVE1728) were cross-mated with the E2F5/p130 double transgenic mice (OVE1811) to generate triple transgenic mice. At E15.5, when compared to nontransgenic FVB lenses (Figure 7A,D), the E2F3a single transgenic lenses showed defects in fiber cell elongation and BrdU positive fiber cells (Figure 7B,E) [15]. The triple transgenic lenses showed less severe pathology (Figure 7C). Although condensed fiber cell nuclei were still present in the center of the triple transgenic lens (Figure 7C), the number of BrdU positive fiber nuclei was decreased by about 50% (p<0.01; Figure 7F; Table 1). Immunohistochemistry showed staining for Myc-tagged E2F3a, E2F5 and p130 proteins in most of the triple transgenic lens fiber cell nuclei (Figure 6C,G,K) when compared to the non-transgenic FVB lenses (Figure 6A,E,I), confirming the expression of these transgenic proteins.


Overexpression of E2F5/p130, but not E2F5 alone, can inhibit E2F-induced cell cycle entry in transgenic mice.

Chen Q, Liang D, Overbeek PA - Mol. Vis. (2008)

E2F3a/E2F5/p130 triple transgenic lens histology and BrdU incorporation assays. A-C: At E15.5, when compared with a non-transgenic FVB lens (A), the triple transgenic lens showed a modest phenotype with some displaced fiber cell nuclei(C) in contrast to the many fiber cell nuclei seen in the E2F3a single transgenic lens (B). Abbreviations: le, lens epithelium; lf, lens fiber. D-F: The number of BrdU positive fiber cells (arrow head) in the triple transgenic lens (F) decreased by about 50% when compared to the E2F3a single transgenic lens (E). Scale bars=500 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f7: E2F3a/E2F5/p130 triple transgenic lens histology and BrdU incorporation assays. A-C: At E15.5, when compared with a non-transgenic FVB lens (A), the triple transgenic lens showed a modest phenotype with some displaced fiber cell nuclei(C) in contrast to the many fiber cell nuclei seen in the E2F3a single transgenic lens (B). Abbreviations: le, lens epithelium; lf, lens fiber. D-F: The number of BrdU positive fiber cells (arrow head) in the triple transgenic lens (F) decreased by about 50% when compared to the E2F3a single transgenic lens (E). Scale bars=500 μm.
Mentions: Hypophosphorylated p130 forms a repressor complex with the E2F4, which can block E2F-responsive promoter sites, and can inhibit the G1/S transition of the cell cycle [18-20]. We hypothesized that the E2F5/p130 complex could also function as a cell cycle repressor in vivo. To examine whether coexpression of E2F5 and p130 can inhibit cell proliferation induced by E2F3a, E2F3a transgenic mice (family OVE1728) were cross-mated with the E2F5/p130 double transgenic mice (OVE1811) to generate triple transgenic mice. At E15.5, when compared to nontransgenic FVB lenses (Figure 7A,D), the E2F3a single transgenic lenses showed defects in fiber cell elongation and BrdU positive fiber cells (Figure 7B,E) [15]. The triple transgenic lenses showed less severe pathology (Figure 7C). Although condensed fiber cell nuclei were still present in the center of the triple transgenic lens (Figure 7C), the number of BrdU positive fiber nuclei was decreased by about 50% (p<0.01; Figure 7F; Table 1). Immunohistochemistry showed staining for Myc-tagged E2F3a, E2F5 and p130 proteins in most of the triple transgenic lens fiber cell nuclei (Figure 6C,G,K) when compared to the non-transgenic FVB lenses (Figure 6A,E,I), confirming the expression of these transgenic proteins.

Bottom Line: However, coexpression of E2F5/p130 with E2F1 or E2F3a in lens fiber cells decreased the number of BrdU positive fiber cells induced by the E2F1 or E2F3a alone.Overexpression of E2F5/p130 in post-mitotic lens fiber cells does not affect their normal differentiation program, but can inhibit inappropriate cell cycle reentry induced by the activator E2Fs.Since E2F5 alone cannot interfere with these E2F activities, we conclude that p130 is a key player in the inhibitory process.

View Article: PubMed Central - PubMed

Affiliation: College of Optometry, University of Houston, Houston, TX 77204-2020, USA.

ABSTRACT

Purpose: The retinoblastoma (Rb) gene family member p130 binds preferentially to the E2F5 transcription factor and forms a repressive E2F5/p130 complex that inhibits cell cycle progression and tumor growth. It is unclear whether E2F5, either alone or in combination with p130, can interfere with the transcriptional activity of other E2F family members, such as E2F1 and E2F3a, in vivo. In this study, we used transgenic mice to test whether overexpression of E2F5 with/without p130 would be sufficient to inhibit E2F1 or E2F3a induced cell cycle reentry.

Methods: Transgenic mice were generated by microinjection of constructs containing different E2F cDNAs (E2F1, E2F3a, and E2F5) or the p130 cDNA linked to the mouse alphaA-crystallin promoter. The E2F5 single and E2F5/p130 double transgenic mice were cross-mated with E2F1 or E2F3a transgenic mice. The resulting double or triple transgenic mouse embryos were characterized by histology, in situ hybridization, immunohistochemistry, and BrdU incorporation assays.

Results: Overexpression of E2F5 alone was not sufficient to inhibit E2F1 or E2F3a induced cell cycle reentry in lens fiber cells. Transgenic mice coexpressing E2F5 and p130 in lens fiber cells did not show lens defects. However, coexpression of E2F5/p130 with E2F1 or E2F3a in lens fiber cells decreased the number of BrdU positive fiber cells induced by the E2F1 or E2F3a alone. Therefore, overexpression of E2F5/p130, but not E2F5 alone, can inhibit activator E2F-mediated cell proliferation in vivo, confirming that p130 plays a critical role in the repressive activity of E2F5/p130 complex.

Conclusions: Overexpression of E2F5/p130 in post-mitotic lens fiber cells does not affect their normal differentiation program, but can inhibit inappropriate cell cycle reentry induced by the activator E2Fs. Since E2F5 alone cannot interfere with these E2F activities, we conclude that p130 is a key player in the inhibitory process.

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Related in: MedlinePlus