Limits...
The CDKN1B-RB1-E2F1 pathway protects mouse spermatogonial stem cells from genomic damage.

Tanaka T, Kanatsu-Shinohara M, Shinohara T - J. Reprod. Dev. (2015)

Bottom Line: This DNA damage is caused by increased E2F1 activity, the depletion of which decreased DNA DSBs caused by Rb1 deficiency.Depletion of Cdkn1a and Bbc3, which were upregulated by Trp53, rescued Rb1-deficient cells from undergoing cell cycle arrest and apoptosis.These results suggest that the CDKN1B-RB1-E2F1 pathway is essential for SSC self-renewal and protects SSCs against genomic damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.

ABSTRACT
Spermatogonial stem cells (SSCs) undergo self-renewal divisions to provide the foundation for spermatogenesis. Although Rb1 deficiency is reportedly essential for SSC self-renewal, its mechanism has remained unknown. Here we report that Rb1 is critical for cell cycle progression and protection of SSCs from DNA double-strand breaks (DSBs). Cultured SSCs depleted of Cdkn1b proliferated poorly and showed diminished expression of CDK4 and RB1, thereby leading to hypophosphorylation of RB1. Rb1 deficiency induced cell cycle arrest and apoptosis in cultured SSCs, which expressed markers for DNA DSBs. This DNA damage is caused by increased E2F1 activity, the depletion of which decreased DNA DSBs caused by Rb1 deficiency. Depletion of Cdkn1a and Bbc3, which were upregulated by Trp53, rescued Rb1-deficient cells from undergoing cell cycle arrest and apoptosis. These results suggest that the CDKN1B-RB1-E2F1 pathway is essential for SSC self-renewal and protects SSCs against genomic damage.

No MeSH data available.


Related in: MedlinePlus

Suppression of the cell cycle by Cdkn1a induction following induction of Rb1 deficiency. (A) Real-time PCR analysis of cell cycle-related genes following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection (n = 3). (B) Western blot analysis of CDKI proteins following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection. The graph shows relative band intensity (n = 3). (C) Cell cycle analysis by Hoechst 33342 following Cdkn1a or Cdkn2a OE (MOI = 10). (D) Increased cell recovery of Rb1 KO GS cells following depletion of Cdkn1a. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Cdkn1a and recovered 1 week after infection (n = 3; MOI = 4). (E) Increased cell recovery of Rb1 KO GS cells following double depletion ofBbc3 and Cdkn1a. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against the indicated genes and recovered 1 week after infection (n = 8; MOI = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4547988&req=5

fig_006: Suppression of the cell cycle by Cdkn1a induction following induction of Rb1 deficiency. (A) Real-time PCR analysis of cell cycle-related genes following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection (n = 3). (B) Western blot analysis of CDKI proteins following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection. The graph shows relative band intensity (n = 3). (C) Cell cycle analysis by Hoechst 33342 following Cdkn1a or Cdkn2a OE (MOI = 10). (D) Increased cell recovery of Rb1 KO GS cells following depletion of Cdkn1a. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Cdkn1a and recovered 1 week after infection (n = 3; MOI = 4). (E) Increased cell recovery of Rb1 KO GS cells following double depletion ofBbc3 and Cdkn1a. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against the indicated genes and recovered 1 week after infection (n = 8; MOI = 4).

Mentions: We next examined cell cycle arrest in Rb1 KO GS cells. When the expression of several cell cycle-related genes was examined using real-time PCR, no apparent changes were observed in cyclin expression (Fig. 6AFig. 6.


The CDKN1B-RB1-E2F1 pathway protects mouse spermatogonial stem cells from genomic damage.

Tanaka T, Kanatsu-Shinohara M, Shinohara T - J. Reprod. Dev. (2015)

Suppression of the cell cycle by Cdkn1a induction following induction of Rb1 deficiency. (A) Real-time PCR analysis of cell cycle-related genes following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection (n = 3). (B) Western blot analysis of CDKI proteins following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection. The graph shows relative band intensity (n = 3). (C) Cell cycle analysis by Hoechst 33342 following Cdkn1a or Cdkn2a OE (MOI = 10). (D) Increased cell recovery of Rb1 KO GS cells following depletion of Cdkn1a. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Cdkn1a and recovered 1 week after infection (n = 3; MOI = 4). (E) Increased cell recovery of Rb1 KO GS cells following double depletion ofBbc3 and Cdkn1a. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against the indicated genes and recovered 1 week after infection (n = 8; MOI = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4547988&req=5

fig_006: Suppression of the cell cycle by Cdkn1a induction following induction of Rb1 deficiency. (A) Real-time PCR analysis of cell cycle-related genes following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection (n = 3). (B) Western blot analysis of CDKI proteins following Rb1 gene deletion. Cells were recovered 2 weeks after AxCANCre transfection. The graph shows relative band intensity (n = 3). (C) Cell cycle analysis by Hoechst 33342 following Cdkn1a or Cdkn2a OE (MOI = 10). (D) Increased cell recovery of Rb1 KO GS cells following depletion of Cdkn1a. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against Cdkn1a and recovered 1 week after infection (n = 3; MOI = 4). (E) Increased cell recovery of Rb1 KO GS cells following double depletion ofBbc3 and Cdkn1a. Cells were transfected with AxCANCre and a lentivirus expressing shRNA against the indicated genes and recovered 1 week after infection (n = 8; MOI = 4).
Mentions: We next examined cell cycle arrest in Rb1 KO GS cells. When the expression of several cell cycle-related genes was examined using real-time PCR, no apparent changes were observed in cyclin expression (Fig. 6AFig. 6.

Bottom Line: This DNA damage is caused by increased E2F1 activity, the depletion of which decreased DNA DSBs caused by Rb1 deficiency.Depletion of Cdkn1a and Bbc3, which were upregulated by Trp53, rescued Rb1-deficient cells from undergoing cell cycle arrest and apoptosis.These results suggest that the CDKN1B-RB1-E2F1 pathway is essential for SSC self-renewal and protects SSCs against genomic damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.

ABSTRACT
Spermatogonial stem cells (SSCs) undergo self-renewal divisions to provide the foundation for spermatogenesis. Although Rb1 deficiency is reportedly essential for SSC self-renewal, its mechanism has remained unknown. Here we report that Rb1 is critical for cell cycle progression and protection of SSCs from DNA double-strand breaks (DSBs). Cultured SSCs depleted of Cdkn1b proliferated poorly and showed diminished expression of CDK4 and RB1, thereby leading to hypophosphorylation of RB1. Rb1 deficiency induced cell cycle arrest and apoptosis in cultured SSCs, which expressed markers for DNA DSBs. This DNA damage is caused by increased E2F1 activity, the depletion of which decreased DNA DSBs caused by Rb1 deficiency. Depletion of Cdkn1a and Bbc3, which were upregulated by Trp53, rescued Rb1-deficient cells from undergoing cell cycle arrest and apoptosis. These results suggest that the CDKN1B-RB1-E2F1 pathway is essential for SSC self-renewal and protects SSCs against genomic damage.

No MeSH data available.


Related in: MedlinePlus