OBJECTIVES: This article is to study the role of G(1)/S regulators in differentiation of pluripotent embryonic cells. MATERIALS AND 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. RESULTS AND 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.

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

Zobrazit více v PubMed

Andäng M, Hjerling‐Leffler J, Moliner A, Lundgren TK, Castelo‐Branco G, Nanou E, Pozas E, Bryja V, Halliez S, Nishimaru H, Wilbertz J, Arenas E, Koltzenburg M, Charnay P, El Manira A, Ibañez CF, Ernfors P (2008) Histone H2AX‐dependent GABAA receptor regulation of stem cell proliferation. Nature 451, 460–464. PubMed

Andrews PW, Przyborski SA, Thomson JA (2001) Embryonal carcinoma cells as embryonic stem cells In: Marshak DR, Gardner RL, Gottlieb D, eds. Stem Cell Biology, pp. 231–265. New York: Cold Spring; Harbor Laboratory Press.

Baldassarre G, Barone MV, Belleti B, Sandomenico C, Bruni P, Spiezia S, Boccia A, Vento MT, Romano A, Pepe S, Fusco A, Viglietto G (1999) Key role of the cyclin‐dependent kinase inhibitor p27kip1 for embryonal carcinoma cell survival and differentiation. Oncogene 18, 6241–6251. PubMed

Baldassarre G, Boccia A, Bruni P, Sandomenico C, Barone MV, Pepe S, Angrisano T, Belletti B, Motti ML, Fusco A, Viglietto G (2000) Retinoic acid induces neuronal differentiation of embryonal carcinoma cells by reducing proteasome‐dependent proteolysis of the cyclin‐dependent inhibitor p27. Cell Growth Differ. 11, 517–526. PubMed

Bartkova J, Lukas J, Muller H, Strauss M, Gusterson B, Bartek J (1995) Abnormal patterns of D‐type cyclin expression and G1 regulation in human head and neck cancer. Cancer Res. 55, 949–956. PubMed

Bartkova J, Zemanova M, Bartek J (1996) Abundance and subcellular localisation of cyclin D3 in human tumours. Int. J. Cancer 65, 323–327. PubMed

Bryja V, Cajanek L, Pachernik J, Hall AC, Horvath V, Dvorak P, Hampl A (2005) Abnormal development of mouse embryoid bodies lacking p27kip1 cell cycle regulator. Stem Cells 23, 965–974. PubMed

Bryja V, Pachernik J, Faldikova L, Krejci P, Pogue R, Nevriva I, Dvorak P, Hampl A (2004a) The role of p27(Kip1) in maintaining the levels of D‐type cyclins in vivo . Biochim. Biophys. Acta 1691, 105–116. PubMed

Bryja V, Pachernik J, Soucek K, Horvath V, Dvorak P, Hampl A (2004b) Increased apoptosis in differentiating p27‐deficient mouse embryonic stem cells. Cell Mol. Life Sci. 61, 1384–1400. PubMed PMC

Chellappan SP, Giordano A, Fisher PB (1998) Role of cyclin‐dependent kinases and their inhibitors in cellular differentiation and development. Curr. Top. Microbiol. Immunol. 227, 57–103. PubMed

Ciemerych MA, Kenney AM, Sicinska E, Kalasczynska I, Bronson RT, Rowitch DH, Gardner H, Sicinski P (2002) Development of mice expressing a single D‐type cyclin. Genes Dev. 16, 3277–3289. PubMed PMC

Ekholm SV, Reed SI (2000) Regulation of G(1) cyclin‐dependent kinases in the mammalian cell cycle. Curr. Opin. Cell Biol. 12, 676–684. PubMed

Faast R, White J, Cartwright P, Crocker L, Sarcevic B, Dalton S (2004) Cdk6‐cyclin D3 activity in murine ES cells is resistant to inhibition by p16INK4a . Oncogene 23, 491–502. PubMed

Fantl V, Stamp G, Andrews A, Rosewell I, Dickson C (1995) Mice lacking cyclin D1 are small and show defects in eye and mammary gland development. Genes Dev. 9, 2364–2372. PubMed

Gao CY, Zelenka P (1997) Cyclins, cyclin‐dependent kinases and differentiation. Bioessays 19, 307–315. PubMed

Gill MR, Slack R, Kiess M, Hamel PA (1998) Regulation of expression and activity of distinct pRB, E2F, D‐type cyclin, and CKI family members during terminal differentiation of P19 cells. Exp. Cell Res. 244, 157–170. PubMed

Glozak MA, Rogers MB (2001) Retinoic acid‐ and bone morphogenetic protein 4‐induced apoptosis in P19 embryonal carcinoma cells requires p27. Exp. Cell Res. 268, 128–138. PubMed

Huard JMT, Forster CC, Carter ML, Sicinski P, Ross ME (1999) Cerebellar histogenesis is disturbed in mice lacking cyclin D2. Development 126, 1927–1935. PubMed

Jirmanova L, Bulavin DV, Fornace AJ Jr (2005) Inhibition of the ATR/Chk1 pathway induces a p38‐dependent S‐phase delay in mouse embryonic stem cells. Cell Cycle 4, 1428–1434. PubMed

Kozar K, Ciemerych MA, Rebel VI, Shigematsu H, Zagozdzon A, Sicinska E, Geng Y, Yu Q, Bhattacharya S, Bronson RT, Akashi K, Sicinski P (2004) Mouse development and cell proliferation in the absence of D‐cyclins. Cell 118, 477–491. PubMed

Krejci P, Bryja V, Pachernik J, Hampl A, Pogue R, Mekikian P, Wilcox WR (2004) FGF2 inhibits proliferation and alters the cartilage‐like phenotype of RCS cells. Exp. Cell Res. 297, 152–164. PubMed

Li Y, Glozak MA, Smith SM, Rogers MB (1999) The expression and activity of D‐type cyclins in F9 embryonal carcinoma cells: modulation of growth by RXR‐selective retinoids. Exp. Cell Res. 253, 372–384. PubMed

Lin J, Jinno S, Okayama H (2001) Cdk6‐cyclin D3 complex evades inhibition by inhibitor proteins and uniquely controls cell's proliferation competence. Oncogene 20, 2000–2009. PubMed

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real‐time quantitative PCR and the 2(‐Delta Delta C(T)) Method. Methods 25, 402–408. PubMed

Malumbres M, Sotillo R, Santamaria D, Galan J, Cerezo A, Ortega S, Dubus P, Barbacid M (2004) Mammalian cells cycle without the D‐type cyclin‐dependent kinases Cdk4 and Cdk6. Cell 118, 493–504. PubMed

Nakayama K, Nakayama K (1998) Cip/Kip cyclin‐dependent kinase inhibitors: brakes of the cell cycle engine during development. Bioessays 20, 1020–1029. PubMed

Pachernik J, Bryja V, Esner M, Kubala L, Dvorak P, Hampl A (2005) Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum. Physiol. Res. 54, 115–122. PubMed

Paternot S, Arsenijevic T, Coulonval K, Bockstaele L, Dumont JE, Roger PP (2006) Distinct specificities of pRb phosphorylation by CDK4 activated by cyclin D1 or cyclin D3: differential involvement in the distinct mitogenic modes of thyroid epithelial cells. Cell Cycle 5, 61–70. PubMed

Poguet A‐L, Legrand C, Feng X, Yen PM, Meltzer P, Samarut J, Flamant F (2003) Microarray analysis of knockout mice identifies cyclin D2 as a possible mediator for the action of thyroid hormone during the postnatal development of the cerebellum. Dev. Biol. 254, 188–199. PubMed

Preclikova H, Bryja V, Pachernik J, Krejci P, Dvorak P, Hampl A (2002) Early cycling‐independent changes to p27, cyclin D2, and cyclin D3 in differentiating mouse embryonal carcinoma cells. Cell Growth Differ. 13, 421–430. PubMed

Ross ME, Carter ML, Lee JH (1996) MN20, a D2 cyclin, is transiently expressed in selected neural populations during embryogenesis. J. Neurosci. 16, 210–219. PubMed PMC

Ross ME, Riskin M (1994) MN20, a D2 cyclin found in brain, is implicated in neural differentiation. J. Neurosci. 14, 6384–6391. PubMed PMC

Savatier P, Lapillonne H, Van Grunsven LA, Rudkin BB, Samarut J (1995) Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up‐regulates D‐type cyclins and cyclin‐dependent kinase inhibitors in mouse embryonic stem cells. Oncogene 12, 309–322. PubMed

Sherr CJ (1995) D‐type cyclins. Trends Biochem. Sci. 20, 187–190. PubMed

Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1‐phase progression. Genes Dev. 13, 1501–1512. PubMed

Sicinski P, Donaher JL, Parker SB, Li T, Fazeli A, Gardner H, Haslam SZ, Bronson RT, Elledge SJ, Weinberg RA (1995) Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 82, 621–630. PubMed

Slee EA, Adrain C, Martin SJ (2001) Executioner caspase‐3‐6, and ‐7 perform distinct, non‐redundant roles during the demolition phase of apoptosis. J. Biol. Chem. 276, 7320–7326. PubMed

Smith A (2001) Embryonic stem cells In: Marshak DR, Gardner RL, Gottlieb D, eds. Stem Cell Biology, pp. 205–230. New York: Cold Spring; Harbor Laboratory Press.

Sumrejkanchanakij P, Tamamori‐Adachi M, Matsunaga Y, Eto K (2003) Role of cyclin D1 cytoplasmic sequestration in the survival of postmitotic neurons. Oncogene 22, 8723–8730. PubMed

Takaki T, Fukasawa K, Suzuki‐Takahashi I, Semba K, Kitagawa M, Taya Y, Hirai H (2005) Preferences for phosphorylation sites in the retinoblastoma protein of D‐type cyclin‐dependent kinases, Cdk4 and Cdk6, in vitro . J. Biochem. (Tokyo) 137, 381–386. PubMed

Watanabe Y, Watanabe T, Kitagawa M, Taya Y, Nakayama K, Motoyama N (1999) pRb phosphorylation is regulated differentially by cyclin‐dependent kinase (Cdk) 2 and Cdk4 in retinoic acid‐induced neuronal differentiation of P19 cells. Brain Res. 842, 342–350. PubMed

Alkaline phosphatase in stem cells

Interaction of Notch and gp130 signaling in the maintenance of neural stem and progenitor cells

Regulatory dephosphorylation of CDK at G₂/M in plants: yeast mitotic phosphatase cdc25 induces cytokinin-like effects in transgenic tobacco morphogenesis