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Genetic characterization of p27(kip1) and stathmin in controlling cell proliferation in vivo.

Berton S, Pellizzari I, Fabris L, D'Andrea S, Segatto I, Canzonieri V, Marconi D, Schiappacassi M, Benevol S, Gattei V, Colombatti A, Belletti B, Baldassarre G - Cell Cycle (2014)

Bottom Line: We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner.In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2.Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

View Article: PubMed Central - PubMed

Affiliation: a Department of Translational Research; Division of Experimental Oncology 2; CRO of Aviano, National Cancer Institute ; Aviano , Italy.

ABSTRACT
The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

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Stathmin loss reverts the outgrowth of the retina basal layer of p27KO mice. (A) H&E staining of the retina in sections from 5-weeks-old WT, p27KO, DKO and StmKO C57BL/6 mice. Black arrows indicate groups of cells outgrowing from the retina basal layer. (B) Graphs report the quantification of retina outgrowth in mice of the indicated genotypes. Only groups of 10 or more cells were counted. The number of analyzed mice is reported in the graph. Statistical significance is calculated by unpaired t-test and expressed by a p value ≤ 0.05. (C) Confocal images of immunofluorescence analysis of stathmin (green) and nuclei (red) in retinal sections from WT, p27KO, DKO and StmKO C57BL/6 5-weeks-old mice. Bottom panels represent the merge of the 2 staining. Pictures show the endogenous expression of stathmin in murine retinas from WT and p27KO animals and its absence in tissue from DKO and StmKO mice, demonstrating the specificity of the immunostaining. Arrows indicate groups of stathmin positive cells outgrowing from the basal layer of the retina. A 40x objective was used.
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f0003: Stathmin loss reverts the outgrowth of the retina basal layer of p27KO mice. (A) H&E staining of the retina in sections from 5-weeks-old WT, p27KO, DKO and StmKO C57BL/6 mice. Black arrows indicate groups of cells outgrowing from the retina basal layer. (B) Graphs report the quantification of retina outgrowth in mice of the indicated genotypes. Only groups of 10 or more cells were counted. The number of analyzed mice is reported in the graph. Statistical significance is calculated by unpaired t-test and expressed by a p value ≤ 0.05. (C) Confocal images of immunofluorescence analysis of stathmin (green) and nuclei (red) in retinal sections from WT, p27KO, DKO and StmKO C57BL/6 5-weeks-old mice. Bottom panels represent the merge of the 2 staining. Pictures show the endogenous expression of stathmin in murine retinas from WT and p27KO animals and its absence in tissue from DKO and StmKO mice, demonstrating the specificity of the immunostaining. Arrows indicate groups of stathmin positive cells outgrowing from the basal layer of the retina. A 40x objective was used.

Mentions: First, we analyzed retinas explanted from 5-8 weeks-old mice. As previously reported, we observed that all p27KO mice displayed outgrowth of the retina basal layer (Fig. 3A and B). In order to exclude possible artifacts due to samples fixing, processing and staining and to obtain a quantification of the outgrowth, we scored the cell groups (more than 10 cells) grown outside the retina basal layer, in n = 10 p27KO mice (20 retinas) (Fig. 3A, arrows). Using this criterion we observed that, on average, p27KO mice presented 2.3 lesions/retina (Fig. 3B). The same analysis was then performed in retinas from mice of the other genotypes. WT and StmKO retinas did not display any lesion and, more interestingly, DKO mice (n = 6 mice, n = 12 retinas) displayed only 0.5 lesions/retina. Immunofluorescence analyses confirmed that stathmin was well expressed in the retina of WT and p27KO mice and that it was clearly visible in the cells grown outside of the basal layer (Fig. 3C, arrows).Figure 3.


Genetic characterization of p27(kip1) and stathmin in controlling cell proliferation in vivo.

Berton S, Pellizzari I, Fabris L, D'Andrea S, Segatto I, Canzonieri V, Marconi D, Schiappacassi M, Benevol S, Gattei V, Colombatti A, Belletti B, Baldassarre G - Cell Cycle (2014)

Stathmin loss reverts the outgrowth of the retina basal layer of p27KO mice. (A) H&E staining of the retina in sections from 5-weeks-old WT, p27KO, DKO and StmKO C57BL/6 mice. Black arrows indicate groups of cells outgrowing from the retina basal layer. (B) Graphs report the quantification of retina outgrowth in mice of the indicated genotypes. Only groups of 10 or more cells were counted. The number of analyzed mice is reported in the graph. Statistical significance is calculated by unpaired t-test and expressed by a p value ≤ 0.05. (C) Confocal images of immunofluorescence analysis of stathmin (green) and nuclei (red) in retinal sections from WT, p27KO, DKO and StmKO C57BL/6 5-weeks-old mice. Bottom panels represent the merge of the 2 staining. Pictures show the endogenous expression of stathmin in murine retinas from WT and p27KO animals and its absence in tissue from DKO and StmKO mice, demonstrating the specificity of the immunostaining. Arrows indicate groups of stathmin positive cells outgrowing from the basal layer of the retina. A 40x objective was used.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4612673&req=5

f0003: Stathmin loss reverts the outgrowth of the retina basal layer of p27KO mice. (A) H&E staining of the retina in sections from 5-weeks-old WT, p27KO, DKO and StmKO C57BL/6 mice. Black arrows indicate groups of cells outgrowing from the retina basal layer. (B) Graphs report the quantification of retina outgrowth in mice of the indicated genotypes. Only groups of 10 or more cells were counted. The number of analyzed mice is reported in the graph. Statistical significance is calculated by unpaired t-test and expressed by a p value ≤ 0.05. (C) Confocal images of immunofluorescence analysis of stathmin (green) and nuclei (red) in retinal sections from WT, p27KO, DKO and StmKO C57BL/6 5-weeks-old mice. Bottom panels represent the merge of the 2 staining. Pictures show the endogenous expression of stathmin in murine retinas from WT and p27KO animals and its absence in tissue from DKO and StmKO mice, demonstrating the specificity of the immunostaining. Arrows indicate groups of stathmin positive cells outgrowing from the basal layer of the retina. A 40x objective was used.
Mentions: First, we analyzed retinas explanted from 5-8 weeks-old mice. As previously reported, we observed that all p27KO mice displayed outgrowth of the retina basal layer (Fig. 3A and B). In order to exclude possible artifacts due to samples fixing, processing and staining and to obtain a quantification of the outgrowth, we scored the cell groups (more than 10 cells) grown outside the retina basal layer, in n = 10 p27KO mice (20 retinas) (Fig. 3A, arrows). Using this criterion we observed that, on average, p27KO mice presented 2.3 lesions/retina (Fig. 3B). The same analysis was then performed in retinas from mice of the other genotypes. WT and StmKO retinas did not display any lesion and, more interestingly, DKO mice (n = 6 mice, n = 12 retinas) displayed only 0.5 lesions/retina. Immunofluorescence analyses confirmed that stathmin was well expressed in the retina of WT and p27KO mice and that it was clearly visible in the cells grown outside of the basal layer (Fig. 3C, arrows).Figure 3.

Bottom Line: We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner.In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2.Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

View Article: PubMed Central - PubMed

Affiliation: a Department of Translational Research; Division of Experimental Oncology 2; CRO of Aviano, National Cancer Institute ; Aviano , Italy.

ABSTRACT
The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

Show MeSH
Related in: MedlinePlus