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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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Generation and characterization of E2F5/p130 double transgenic mice. A: Microinjected constructs. Human E2F5 and mouse p130 cDNAs were linked to the δenhancer αA-crystallin promoter in vector δenαA-minx (DREAM) [30]. B: At E15.5, the bigenic lens shows a normal lens phenotype. C: No BrdU positive fiber cells were present in the center of the double transgenic lens. D-E: In situ hybridization was used to examine expression of E2F5 and p130 transgenes with human E2F5 and mouse p130 riboprobes. Hybridization signals were initially captured as dark-field images, pseudocolored red, and then superimposed on bright-field images of the same tissue sections counterstained by hematoxylin. The E2F5 (D) and p130 (E) transgenes were expressed in lens fiber cells. The size of lens in panel E is smaller as this section is more peripheral. F-G: Immunohistochemistry. Using antibodies against E2F5 (F) and p130 (G), a green nuclear staining was detected in the double transgenic lens fiber cells. Scale bars=500 μm.
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f1: Generation and characterization of E2F5/p130 double transgenic mice. A: Microinjected constructs. Human E2F5 and mouse p130 cDNAs were linked to the δenhancer αA-crystallin promoter in vector δenαA-minx (DREAM) [30]. B: At E15.5, the bigenic lens shows a normal lens phenotype. C: No BrdU positive fiber cells were present in the center of the double transgenic lens. D-E: In situ hybridization was used to examine expression of E2F5 and p130 transgenes with human E2F5 and mouse p130 riboprobes. Hybridization signals were initially captured as dark-field images, pseudocolored red, and then superimposed on bright-field images of the same tissue sections counterstained by hematoxylin. The E2F5 (D) and p130 (E) transgenes were expressed in lens fiber cells. The size of lens in panel E is smaller as this section is more peripheral. F-G: Immunohistochemistry. Using antibodies against E2F5 (F) and p130 (G), a green nuclear staining was detected in the double transgenic lens fiber cells. Scale bars=500 μm.

Mentions: All animals were used in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Human E2F1, Myc-tagged mouse E2F3a and human E2F5 transgenic lines were generated as previously described [15,29]. E2F5/p130 double transgenic mice were generated by coinjection. A Sac II – Xba I fragment containing the human E2F5 cDNA and a Cla I – EcoR V fragment carrying mouse p130 cDNA were cloned downstream from the δenhancer/αA-crystallin promoter in the vector δenαA-minx (DREAM) [30]. The resultant plasmids (Figure 1A) were digested with KpnI and NotI to release fragments for microinjection. The fragments were separated by electrophoresis through a 1.2% agarose gel and purified using a Qiaex II gel extraction kit (Qiagen, Hilden, Germany). Transgenic mice were generated by pronuclear coinjection of both fragments into one-cell-stage inbred FVB/N embryos [31,32]. Two stable transgenic families (OVE1811 and OVE1812) expressing both E2F5 and p130 transgenes were generated from the coinjection. E2F5 single (OVE1598) and E2F5/p130 double (OVE1811) transgenic mice were cross-mated with E2F1 (OVE527) or E2F3a (OVE1728) transgenic mice to generate double (E2F5/E2F1 or E2F5/E2F3a) and triple (E2F5/p130/E2F1 or E2F5/p130/E2F3a) transgenic mice.


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)

Generation and characterization of E2F5/p130 double transgenic mice. A: Microinjected constructs. Human E2F5 and mouse p130 cDNAs were linked to the δenhancer αA-crystallin promoter in vector δenαA-minx (DREAM) [30]. B: At E15.5, the bigenic lens shows a normal lens phenotype. C: No BrdU positive fiber cells were present in the center of the double transgenic lens. D-E: In situ hybridization was used to examine expression of E2F5 and p130 transgenes with human E2F5 and mouse p130 riboprobes. Hybridization signals were initially captured as dark-field images, pseudocolored red, and then superimposed on bright-field images of the same tissue sections counterstained by hematoxylin. The E2F5 (D) and p130 (E) transgenes were expressed in lens fiber cells. The size of lens in panel E is smaller as this section is more peripheral. F-G: Immunohistochemistry. Using antibodies against E2F5 (F) and p130 (G), a green nuclear staining was detected in the double transgenic lens fiber cells. Scale bars=500 μm.
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f1: Generation and characterization of E2F5/p130 double transgenic mice. A: Microinjected constructs. Human E2F5 and mouse p130 cDNAs were linked to the δenhancer αA-crystallin promoter in vector δenαA-minx (DREAM) [30]. B: At E15.5, the bigenic lens shows a normal lens phenotype. C: No BrdU positive fiber cells were present in the center of the double transgenic lens. D-E: In situ hybridization was used to examine expression of E2F5 and p130 transgenes with human E2F5 and mouse p130 riboprobes. Hybridization signals were initially captured as dark-field images, pseudocolored red, and then superimposed on bright-field images of the same tissue sections counterstained by hematoxylin. The E2F5 (D) and p130 (E) transgenes were expressed in lens fiber cells. The size of lens in panel E is smaller as this section is more peripheral. F-G: Immunohistochemistry. Using antibodies against E2F5 (F) and p130 (G), a green nuclear staining was detected in the double transgenic lens fiber cells. Scale bars=500 μm.
Mentions: All animals were used in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Human E2F1, Myc-tagged mouse E2F3a and human E2F5 transgenic lines were generated as previously described [15,29]. E2F5/p130 double transgenic mice were generated by coinjection. A Sac II – Xba I fragment containing the human E2F5 cDNA and a Cla I – EcoR V fragment carrying mouse p130 cDNA were cloned downstream from the δenhancer/αA-crystallin promoter in the vector δenαA-minx (DREAM) [30]. The resultant plasmids (Figure 1A) were digested with KpnI and NotI to release fragments for microinjection. The fragments were separated by electrophoresis through a 1.2% agarose gel and purified using a Qiaex II gel extraction kit (Qiagen, Hilden, Germany). Transgenic mice were generated by pronuclear coinjection of both fragments into one-cell-stage inbred FVB/N embryos [31,32]. Two stable transgenic families (OVE1811 and OVE1812) expressing both E2F5 and p130 transgenes were generated from the coinjection. E2F5 single (OVE1598) and E2F5/p130 double (OVE1811) transgenic mice were cross-mated with E2F1 (OVE527) or E2F3a (OVE1728) transgenic mice to generate double (E2F5/E2F1 or E2F5/E2F3a) and triple (E2F5/p130/E2F1 or E2F5/p130/E2F3a) transgenic mice.

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.

Show MeSH
Related in: MedlinePlus