Limits...
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.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.

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

Stathmin loss reverts the increased organ size of p27KO mice. (A) Graphs report the organ/body ratio of spleens (upper panel) and thymuses (lower panel) from WT, p27KO, DKO and StmKO 15-weeks-old C57BL/6 mice. Each dot corresponds to one mouse. (B) Graphs report the weight (upper panel) and number of cells/organ (lower panel) in spleens and thymuses from WT, p27KO, DKO and StmKO 10-weeks-old C57BL/6 mice. (C) Western Blot analysis of p27 and stathmin expression in thymus, spleen and brain lysates, obtained from 2 WT, 2 p27KO and one DKO C57BL/6 mice, as indicated. Vinculin was used as loading control. Asterisk indicates non-specific bands detected by anti-p27 antibody. (D) Graphs report the weights of thymus, spleen, testis and brain from mice of the indicated genotypes. Each dot corresponds to one mouse. In each graph, statistical significance is calculated by unpaired t-test and expressed by a p value ≤ 0.05 (ns, not significant).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4612673&req=5

f0002: Stathmin loss reverts the increased organ size of p27KO mice. (A) Graphs report the organ/body ratio of spleens (upper panel) and thymuses (lower panel) from WT, p27KO, DKO and StmKO 15-weeks-old C57BL/6 mice. Each dot corresponds to one mouse. (B) Graphs report the weight (upper panel) and number of cells/organ (lower panel) in spleens and thymuses from WT, p27KO, DKO and StmKO 10-weeks-old C57BL/6 mice. (C) Western Blot analysis of p27 and stathmin expression in thymus, spleen and brain lysates, obtained from 2 WT, 2 p27KO and one DKO C57BL/6 mice, as indicated. Vinculin was used as loading control. Asterisk indicates non-specific bands detected by anti-p27 antibody. (D) Graphs report the weights of thymus, spleen, testis and brain from mice of the indicated genotypes. Each dot corresponds to one mouse. In each graph, statistical significance is calculated by unpaired t-test and expressed by a p value ≤ 0.05 (ns, not significant).

Mentions: Since knock-out of p27 gene leads to particular enlargement of lymphoid organs,8-10 we first focused our analyses on thymus and spleen. By calculating the ratio between organ/body weights of 15 weeks old mice, we observed a significant reduction in DKO mice when compared to p27KO animals (Fig. 2A). It has been demonstrated that the enlargement of p27KO lymphoid organs is due to higher cell number.8-10 Accordingly, analyses of 10 weeks old males (n = 3/group) for weight and cell number in lymphoid organs indicated the presence of higher numbers of both splenocytes and thymocytes in p27KO respect to DKO and WT mice (Fig. 2B). The differences between p27KO and the other genotypes were particularly evident for thymocytes, which were highly and more significantly reduced in DKO animals (Fig. 2B). This observation prompted us to verify if the difference in organ weight directly correlated with the expression of stathmin in adult organs. To this aim, we looked at the weights of the 3 organs in which stathmin was most highly expressed, namely the brain, the thymus and the testis, and of a organ displaying lower stathmin expression, such as the spleen (Figs. 1 and 2C). Interestingly, the levels of stathmin inversely correlated with organ weights (Fig. 2D). Organs expressing high levels of stathmin displayed a more evident and significant weight reduction in DKO mice respect to p27KO ones and not statistically significant differences between DKO, WT and StmKO mice were detected (Fig. 2D). Conversely, spleens, which express very low levels of stathmin, displayed an intermediate weight in DKO mice respect to p27KO and both WT and StmKO mice. These data were further confirmed in p27 heterozygous and knock out mice, in which stathmin biallelic deletion significantly reduced the weight of all analyzed organs, except for spleens (Fig. S5). These findings reinforce the hypothesis that, when abundantly expressed, stathmin participated with p27 to the control of mouse and organ growth.Figure 2.


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 increased organ size of p27KO mice. (A) Graphs report the organ/body ratio of spleens (upper panel) and thymuses (lower panel) from WT, p27KO, DKO and StmKO 15-weeks-old C57BL/6 mice. Each dot corresponds to one mouse. (B) Graphs report the weight (upper panel) and number of cells/organ (lower panel) in spleens and thymuses from WT, p27KO, DKO and StmKO 10-weeks-old C57BL/6 mice. (C) Western Blot analysis of p27 and stathmin expression in thymus, spleen and brain lysates, obtained from 2 WT, 2 p27KO and one DKO C57BL/6 mice, as indicated. Vinculin was used as loading control. Asterisk indicates non-specific bands detected by anti-p27 antibody. (D) Graphs report the weights of thymus, spleen, testis and brain from mice of the indicated genotypes. Each dot corresponds to one mouse. In each graph, statistical significance is calculated by unpaired t-test and expressed by a p value ≤ 0.05 (ns, not significant).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0002: Stathmin loss reverts the increased organ size of p27KO mice. (A) Graphs report the organ/body ratio of spleens (upper panel) and thymuses (lower panel) from WT, p27KO, DKO and StmKO 15-weeks-old C57BL/6 mice. Each dot corresponds to one mouse. (B) Graphs report the weight (upper panel) and number of cells/organ (lower panel) in spleens and thymuses from WT, p27KO, DKO and StmKO 10-weeks-old C57BL/6 mice. (C) Western Blot analysis of p27 and stathmin expression in thymus, spleen and brain lysates, obtained from 2 WT, 2 p27KO and one DKO C57BL/6 mice, as indicated. Vinculin was used as loading control. Asterisk indicates non-specific bands detected by anti-p27 antibody. (D) Graphs report the weights of thymus, spleen, testis and brain from mice of the indicated genotypes. Each dot corresponds to one mouse. In each graph, statistical significance is calculated by unpaired t-test and expressed by a p value ≤ 0.05 (ns, not significant).
Mentions: Since knock-out of p27 gene leads to particular enlargement of lymphoid organs,8-10 we first focused our analyses on thymus and spleen. By calculating the ratio between organ/body weights of 15 weeks old mice, we observed a significant reduction in DKO mice when compared to p27KO animals (Fig. 2A). It has been demonstrated that the enlargement of p27KO lymphoid organs is due to higher cell number.8-10 Accordingly, analyses of 10 weeks old males (n = 3/group) for weight and cell number in lymphoid organs indicated the presence of higher numbers of both splenocytes and thymocytes in p27KO respect to DKO and WT mice (Fig. 2B). The differences between p27KO and the other genotypes were particularly evident for thymocytes, which were highly and more significantly reduced in DKO animals (Fig. 2B). This observation prompted us to verify if the difference in organ weight directly correlated with the expression of stathmin in adult organs. To this aim, we looked at the weights of the 3 organs in which stathmin was most highly expressed, namely the brain, the thymus and the testis, and of a organ displaying lower stathmin expression, such as the spleen (Figs. 1 and 2C). Interestingly, the levels of stathmin inversely correlated with organ weights (Fig. 2D). Organs expressing high levels of stathmin displayed a more evident and significant weight reduction in DKO mice respect to p27KO ones and not statistically significant differences between DKO, WT and StmKO mice were detected (Fig. 2D). Conversely, spleens, which express very low levels of stathmin, displayed an intermediate weight in DKO mice respect to p27KO and both WT and StmKO mice. These data were further confirmed in p27 heterozygous and knock out mice, in which stathmin biallelic deletion significantly reduced the weight of all analyzed organs, except for spleens (Fig. S5). These findings reinforce the hypothesis that, when abundantly expressed, stathmin participated with p27 to the control of mouse and organ growth.Figure 2.

Bottom Line: We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner.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.

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