Cell cycle block at G1-S or G2-M phase correlates with differentiation of Caco-2 cells: effect of constitutive insulin-like growth factor II expression.
OBJECTIVE
We have previously shown that autocrine insulin-like growth factor (IGF)-II synthesis through IGF-I receptor stimulates proliferation and inhibits differentiation of Caco-2 cells. To demonstrate whether differentiation of Caco-2 cells is dependent on cell growth status, we analyzed the effect of cell cycle arrest on differentiation of wild-type and IGF-II-overexpressing cells.


METHODS
Cells were treated with drugs that inhibit the progression either to S phase (l-b-D-arabinofuranosylcytosine or M phase (nocodazole). Cell differentiation was analyzed by assessing apolipoprotein A-1 and sucrase-isomaltase expression. Cell proliferation and DNA content were assessed by thymidine incorporation and fluorescence-activated cell sorter analysis, respectively. Cell cycle regulatory molecules were analyzed by assessing p21 and retinoplasma protein (pRb) expression and pRb phosphorylation.


RESULTS
Cell cycle block at G1-S phase was associated with increased expression of differentiation markers in both parental and IGF-II-transfected cells. On the contrary, cell cycle arrest at G2-M phase correlated with the expression of differentiation markers in parental but not in IGF-II-transfected cells. Constitutive IGF-II-expressing cells actively incorporated thymidine and showed an increase in the proportion of cells with >4N DNA ploidy in the presence of nocodazole. Nocodazole treatment of constitutive IGF-II-expressing cells stimulated p21 expression in the presence of hyperphosphorylated pRb.


CONCLUSIONS
The data show that cell cycle arrest increases differentiation of Caco-2 cells. IGF-II-mediated proliferation may prevent cell differentiation through effects on control cell checkpoint proteins.
Zarrilli
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Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche "G. Salvatore," Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano, Naples, Italy.
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D000900:Anti-Bacterial Agents / Q000494:pharmacology; D018938:Caco-2 Cells; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D003561:Cytarabine / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics; D016193:G1 Phase / Q000502:physiology*; D016195:G2 Phase / Q000502:physiology*; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000166:cytology* / Q000378:metabolism*; D009355:Neomycin / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology; D011003:Ploidies; D016160:Retinoblastoma Protein / Q000378:metabolism; D014162:Transfection
D000900:Anti-Bacterial Agents; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016160:Retinoblastoma Protein; D003561:Cytarabine; D007335:Insulin-Like Growth Factor II; D009355:Neomycin; D015739:Nocodazole