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Normal proliferation and tumorigenesis but impaired pancreatic function in mice lacking the cell cycle regulator sei1.

Fernandez-Marcos PJ, Pantoja C, Gonzalez-Rodriguez A, Martin N, Flores JM, Valverde AM, Hara E, Serrano M - PLoS ONE (2010)

Bottom Line: Sei1- fibroblasts did not show abnormalities regarding proliferation or susceptibility to neoplastic transformation, nor did we observe defects on Cdk4 complexes or E2f activity.These defects were associated to nuclear accumulation of the cell-cycle inhibitors p21(Cip1) and p27(Kip1) in islet cells.We conclude that Sei1 plays an important role in pancreatic beta-cells, which supports a functional link between Sei1 and the core cell cycle regulators specifically in the context of the pancreas.

View Article: PubMed Central - PubMed

Affiliation: Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

ABSTRACT
Sei1 is a positive regulator of proliferation that promotes the assembly of Cdk4-cyclin D complexes and enhances the transcriptional activity of E2f1. The potential oncogenic role of Sei1 is further suggested by its overexpression in various types of human cancers. To study the role of Sei1, we have generated a mouse line deficient for this gene. Sei1- fibroblasts did not show abnormalities regarding proliferation or susceptibility to neoplastic transformation, nor did we observe defects on Cdk4 complexes or E2f activity. Sei1- mice were viable, did not present overt pathologies, had a normal lifespan, and had a normal susceptibility to spontaneous and chemically-induced cancer. Pancreatic insulin-producing cells are known to be particularly sensitive to Cdk4-cyclin D and E2f activities, and we have observed that Sei1 is highly expressed in pancreatic islets compared to other tissues. Interestingly, Sei1- mice present lower number of islets, decreased beta-cell area, impaired insulin secretion, and glucose intolerance. These defects were associated to nuclear accumulation of the cell-cycle inhibitors p21(Cip1) and p27(Kip1) in islet cells. We conclude that Sei1 plays an important role in pancreatic beta-cells, which supports a functional link between Sei1 and the core cell cycle regulators specifically in the context of the pancreas.

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Proliferation, immortalization and transformation of Sei1- MEFs.(A) Growth curves of primary MEFs grown at the indicated serum concentrations. (B) Senescence and immortalization were evaluated performing a 3T3 serial passage protocol of MEFs of the indicated genotype. ΔPDL, increase in population doubling level (C) Colony formation assay measuring the immortalization potential of MEFs of the indicated genotypes. (D) Foci formation assay of MEFs transduced with HRasV12 or with bicistronic E1A/HRasV12. In all the above assays, at least three independent preparations of primary MEFs per genotype were used. Values correspond to average and s.d.
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pone-0008744-g004: Proliferation, immortalization and transformation of Sei1- MEFs.(A) Growth curves of primary MEFs grown at the indicated serum concentrations. (B) Senescence and immortalization were evaluated performing a 3T3 serial passage protocol of MEFs of the indicated genotype. ΔPDL, increase in population doubling level (C) Colony formation assay measuring the immortalization potential of MEFs of the indicated genotypes. (D) Foci formation assay of MEFs transduced with HRasV12 or with bicistronic E1A/HRasV12. In all the above assays, at least three independent preparations of primary MEFs per genotype were used. Values correspond to average and s.d.

Mentions: Cell cycle regulators may have profound effects on the susceptibility of cells to acquire oncogenic properties [1]. Thus, we studied the serum dependence, immortalization and transformation capacity of MEFs deficient for Sei1. As shown in Fig. 4A, Sei1- MEFs proliferate at the same rate as wt MEFs under different serum concentrations. When subjected to the 3T3 serial passage protocol, we could not observe differences in the onset of senescence or in the immortalization of wt and Sei1- MEFs (Fig. 4B). To further test this under more stringent conditions, we seeded wt and Sei1- cells at low cellular density and checked for the appearance of colonies. Sei1- MEFs showed the same low colony formation capacity as wt MEFs, while Ink4a/Arf-double MEFs, used here as a positive control, formed colonies efficiently (Fig. 4C). Finally, we examined the impact of Sei1 deficiency on the susceptibility of MEFs to neoplastic transformation by HRasG12V or by the combination of HRasG12V and E1A. We measured the transformation efficiency by plating infected cells at confluent density and counting the emergence of foci after two weeks in culture. We could not observe differences between Sei1- and wt MEFs when subjected to these oncogenic transformation assays (Fig. 4D). From these data we conclude that the absence of Sei1 has no significant impact on the requirement for external growth factors, ability to immortalize spontaneously, or susceptibility to be oncogenically transformed.


Normal proliferation and tumorigenesis but impaired pancreatic function in mice lacking the cell cycle regulator sei1.

Fernandez-Marcos PJ, Pantoja C, Gonzalez-Rodriguez A, Martin N, Flores JM, Valverde AM, Hara E, Serrano M - PLoS ONE (2010)

Proliferation, immortalization and transformation of Sei1- MEFs.(A) Growth curves of primary MEFs grown at the indicated serum concentrations. (B) Senescence and immortalization were evaluated performing a 3T3 serial passage protocol of MEFs of the indicated genotype. ΔPDL, increase in population doubling level (C) Colony formation assay measuring the immortalization potential of MEFs of the indicated genotypes. (D) Foci formation assay of MEFs transduced with HRasV12 or with bicistronic E1A/HRasV12. In all the above assays, at least three independent preparations of primary MEFs per genotype were used. Values correspond to average and s.d.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2807453&req=5

pone-0008744-g004: Proliferation, immortalization and transformation of Sei1- MEFs.(A) Growth curves of primary MEFs grown at the indicated serum concentrations. (B) Senescence and immortalization were evaluated performing a 3T3 serial passage protocol of MEFs of the indicated genotype. ΔPDL, increase in population doubling level (C) Colony formation assay measuring the immortalization potential of MEFs of the indicated genotypes. (D) Foci formation assay of MEFs transduced with HRasV12 or with bicistronic E1A/HRasV12. In all the above assays, at least three independent preparations of primary MEFs per genotype were used. Values correspond to average and s.d.
Mentions: Cell cycle regulators may have profound effects on the susceptibility of cells to acquire oncogenic properties [1]. Thus, we studied the serum dependence, immortalization and transformation capacity of MEFs deficient for Sei1. As shown in Fig. 4A, Sei1- MEFs proliferate at the same rate as wt MEFs under different serum concentrations. When subjected to the 3T3 serial passage protocol, we could not observe differences in the onset of senescence or in the immortalization of wt and Sei1- MEFs (Fig. 4B). To further test this under more stringent conditions, we seeded wt and Sei1- cells at low cellular density and checked for the appearance of colonies. Sei1- MEFs showed the same low colony formation capacity as wt MEFs, while Ink4a/Arf-double MEFs, used here as a positive control, formed colonies efficiently (Fig. 4C). Finally, we examined the impact of Sei1 deficiency on the susceptibility of MEFs to neoplastic transformation by HRasG12V or by the combination of HRasG12V and E1A. We measured the transformation efficiency by plating infected cells at confluent density and counting the emergence of foci after two weeks in culture. We could not observe differences between Sei1- and wt MEFs when subjected to these oncogenic transformation assays (Fig. 4D). From these data we conclude that the absence of Sei1 has no significant impact on the requirement for external growth factors, ability to immortalize spontaneously, or susceptibility to be oncogenically transformed.

Bottom Line: Sei1- fibroblasts did not show abnormalities regarding proliferation or susceptibility to neoplastic transformation, nor did we observe defects on Cdk4 complexes or E2f activity.These defects were associated to nuclear accumulation of the cell-cycle inhibitors p21(Cip1) and p27(Kip1) in islet cells.We conclude that Sei1 plays an important role in pancreatic beta-cells, which supports a functional link between Sei1 and the core cell cycle regulators specifically in the context of the pancreas.

View Article: PubMed Central - PubMed

Affiliation: Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

ABSTRACT
Sei1 is a positive regulator of proliferation that promotes the assembly of Cdk4-cyclin D complexes and enhances the transcriptional activity of E2f1. The potential oncogenic role of Sei1 is further suggested by its overexpression in various types of human cancers. To study the role of Sei1, we have generated a mouse line deficient for this gene. Sei1- fibroblasts did not show abnormalities regarding proliferation or susceptibility to neoplastic transformation, nor did we observe defects on Cdk4 complexes or E2f activity. Sei1- mice were viable, did not present overt pathologies, had a normal lifespan, and had a normal susceptibility to spontaneous and chemically-induced cancer. Pancreatic insulin-producing cells are known to be particularly sensitive to Cdk4-cyclin D and E2f activities, and we have observed that Sei1 is highly expressed in pancreatic islets compared to other tissues. Interestingly, Sei1- mice present lower number of islets, decreased beta-cell area, impaired insulin secretion, and glucose intolerance. These defects were associated to nuclear accumulation of the cell-cycle inhibitors p21(Cip1) and p27(Kip1) in islet cells. We conclude that Sei1 plays an important role in pancreatic beta-cells, which supports a functional link between Sei1 and the core cell cycle regulators specifically in the context of the pancreas.

Show MeSH
Related in: MedlinePlus