Lineage specific composition of cyclin D-CDK4/CDK6-p27 complexes reveals distinct functions of CDK4, CDK6 and individual D-type cyclins in differentiating cells of embryonic origin.
OBJECTIVE
This article is to study the role of G(1)/S regulators in differentiation of pluripotent embryonic cells.


METHODS
We established a P19 embryonal carcinoma cell-based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G(1)/S regulators were analysed with respect to growth and differentiation parameters of the cells.


CONCLUSIONS
We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E-CDK2 but not of cyclin D-CDK4/6-p27 complexes. In an exponentially growing P19 cell population, the cyclin D1-CDK4 complex is detected, which is replaced by cyclin D2/3-CDK4/6-p27 complex following density arrest. During endodermal differentiation kinase-inactive cyclin D2/D3-CDK4-p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2-CDK4-p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D-CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G(1)/S transition-regulating machinery in early embryonic cells.
Bryja
V
V
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Pacherník
J
J
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Vondráček
J
J
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Souček
K
K
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Čajánek
L
L
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Horvath
V
V
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Holubcová
Z
Z
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Dvořák
P
P
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Hampl
A
A
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
D000818:Animals; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D019070:Cell Lineage; D049109:Cell Proliferation; D019925:Cyclin A / Q000378:metabolism; D056741:Cyclin D; D019927:Cyclin E / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism*; D051361:Cyclin-Dependent Kinase 6 / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism; D016193:G1 Phase; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D011485:Protein Binding; D021381:Protein Transport; D016196:S Phase
D019925:Cyclin A; D056741:Cyclin D; D019927:Cyclin E; D016213:Cyclins; D050760:Cyclin-Dependent Kinase Inhibitor p27; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6