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Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma.

Thomas DM, Johnson SA, Sims NA, Trivett MK, Slavin JL, Rubin BP, Waring P, McArthur GA, Walkley CR, Holloway AJ, Diyagama D, Grim JE, Clurman BE, Bowtell DD, Lee JS, Gutierrez GM, Piscopo DM, Carty SA, Hinds PW - J. Cell Biol. (2004)

Bottom Line: Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts.Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas.Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: Ian Potter Foundation Centre for Cancer Genomics and Predictive Medicine, and Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Center, Victoria, Melbourne, Australia. david.thomas@petermac.org

ABSTRACT
The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

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p27KIP1 is required for terminal growth arrest in vitro. MEFs were cultured in the presence or absence of 100 ng/ml BMP2, ascorbic acid, and β-glycerophosphate for 14 d after confluence. Cells were then passaged. (A) 5 × 104 cells were grown under standard culture conditions for 3 d, and then counted. Data shown are means ± SEM. (B) RT-qPCR analysis of osteocalcin gene expression in MEFs of the indicated genotypes 2 d after passage. Data shown are means ± SEM of cycle number (ΔΔCT) normalized to ARPPo and expression before passage. The interaction between BMP2 and genotype was significant (P < 0.01, ANOVA). (C) Photomicrograph of senescence-associated β-galactosidase–stained cultures. (i) Wild-type untreated cultures; (ii) p27KIP1−/− untreated cultures; (iii) wild-type differentiated cultures; and (iv) p27KIP1−/− differentiated cultures. (D) Cells were stained for senescence-associated (SA) β-galactosidase activity after passage and counted (>200 cells in triplicate cultures). Data shown are means ± SEM. The effect of BMP2 and genotype was significant (P < 0.01), although there was no interaction by ANOVA.
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fig6: p27KIP1 is required for terminal growth arrest in vitro. MEFs were cultured in the presence or absence of 100 ng/ml BMP2, ascorbic acid, and β-glycerophosphate for 14 d after confluence. Cells were then passaged. (A) 5 × 104 cells were grown under standard culture conditions for 3 d, and then counted. Data shown are means ± SEM. (B) RT-qPCR analysis of osteocalcin gene expression in MEFs of the indicated genotypes 2 d after passage. Data shown are means ± SEM of cycle number (ΔΔCT) normalized to ARPPo and expression before passage. The interaction between BMP2 and genotype was significant (P < 0.01, ANOVA). (C) Photomicrograph of senescence-associated β-galactosidase–stained cultures. (i) Wild-type untreated cultures; (ii) p27KIP1−/− untreated cultures; (iii) wild-type differentiated cultures; and (iv) p27KIP1−/− differentiated cultures. (D) Cells were stained for senescence-associated (SA) β-galactosidase activity after passage and counted (>200 cells in triplicate cultures). Data shown are means ± SEM. The effect of BMP2 and genotype was significant (P < 0.01), although there was no interaction by ANOVA.

Mentions: p27KIP1 plays a key role in terminal cell cycle exit in osteosarcoma cells (Alexander and Hinds, 2001). Because terminal differentiation is associated with permanent cell cycle withdrawal, we tested whether MEF cultures lacking p27KIP1 could reenter the cell cycle after culture under prolonged differentiating conditions. There was no prior difference in cell cycle profile of littermate wild-type or p27KIP1−/− preconfluent early passage MEFs (unpublished data). However, although wild-type MEFs postdifferentiation grew poorly after passage, MEFs lacking p27KIP1 proliferated robustly (Fig. 6 A). This suggests a role for p27KIP1 in mediating the irreversibility of the postconfluent state. However, wild-type undifferentiated MEFs also failed to reenter the cell cycle, probably because p27KIP1 also accumulated in untreated cultures upon confluence (Hirano et al., 2001; and unpublished data). Consistent with an additional effect of BMP2 upon p27KIP1, the degree of growth inhibition after passage was greater in cultures treated with BMP2 (Fig. 6 A). Moreover, even postdifferentiated MEFs lacking p27KIP1 demonstrated a reduction in cell growth when passaged after BMP2 treatment, indicating that p27KIP1 contributes to (but is not solely responsible for) terminal growth arrest. We have not observed compensatory increases in p21CIP1 or p57KIP2 in p27KIP1- cultures (unpublished data), and the mechanisms accounting for the residual effects are not known.


Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarcoma.

Thomas DM, Johnson SA, Sims NA, Trivett MK, Slavin JL, Rubin BP, Waring P, McArthur GA, Walkley CR, Holloway AJ, Diyagama D, Grim JE, Clurman BE, Bowtell DD, Lee JS, Gutierrez GM, Piscopo DM, Carty SA, Hinds PW - J. Cell Biol. (2004)

p27KIP1 is required for terminal growth arrest in vitro. MEFs were cultured in the presence or absence of 100 ng/ml BMP2, ascorbic acid, and β-glycerophosphate for 14 d after confluence. Cells were then passaged. (A) 5 × 104 cells were grown under standard culture conditions for 3 d, and then counted. Data shown are means ± SEM. (B) RT-qPCR analysis of osteocalcin gene expression in MEFs of the indicated genotypes 2 d after passage. Data shown are means ± SEM of cycle number (ΔΔCT) normalized to ARPPo and expression before passage. The interaction between BMP2 and genotype was significant (P < 0.01, ANOVA). (C) Photomicrograph of senescence-associated β-galactosidase–stained cultures. (i) Wild-type untreated cultures; (ii) p27KIP1−/− untreated cultures; (iii) wild-type differentiated cultures; and (iv) p27KIP1−/− differentiated cultures. (D) Cells were stained for senescence-associated (SA) β-galactosidase activity after passage and counted (>200 cells in triplicate cultures). Data shown are means ± SEM. The effect of BMP2 and genotype was significant (P < 0.01), although there was no interaction by ANOVA.
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fig6: p27KIP1 is required for terminal growth arrest in vitro. MEFs were cultured in the presence or absence of 100 ng/ml BMP2, ascorbic acid, and β-glycerophosphate for 14 d after confluence. Cells were then passaged. (A) 5 × 104 cells were grown under standard culture conditions for 3 d, and then counted. Data shown are means ± SEM. (B) RT-qPCR analysis of osteocalcin gene expression in MEFs of the indicated genotypes 2 d after passage. Data shown are means ± SEM of cycle number (ΔΔCT) normalized to ARPPo and expression before passage. The interaction between BMP2 and genotype was significant (P < 0.01, ANOVA). (C) Photomicrograph of senescence-associated β-galactosidase–stained cultures. (i) Wild-type untreated cultures; (ii) p27KIP1−/− untreated cultures; (iii) wild-type differentiated cultures; and (iv) p27KIP1−/− differentiated cultures. (D) Cells were stained for senescence-associated (SA) β-galactosidase activity after passage and counted (>200 cells in triplicate cultures). Data shown are means ± SEM. The effect of BMP2 and genotype was significant (P < 0.01), although there was no interaction by ANOVA.
Mentions: p27KIP1 plays a key role in terminal cell cycle exit in osteosarcoma cells (Alexander and Hinds, 2001). Because terminal differentiation is associated with permanent cell cycle withdrawal, we tested whether MEF cultures lacking p27KIP1 could reenter the cell cycle after culture under prolonged differentiating conditions. There was no prior difference in cell cycle profile of littermate wild-type or p27KIP1−/− preconfluent early passage MEFs (unpublished data). However, although wild-type MEFs postdifferentiation grew poorly after passage, MEFs lacking p27KIP1 proliferated robustly (Fig. 6 A). This suggests a role for p27KIP1 in mediating the irreversibility of the postconfluent state. However, wild-type undifferentiated MEFs also failed to reenter the cell cycle, probably because p27KIP1 also accumulated in untreated cultures upon confluence (Hirano et al., 2001; and unpublished data). Consistent with an additional effect of BMP2 upon p27KIP1, the degree of growth inhibition after passage was greater in cultures treated with BMP2 (Fig. 6 A). Moreover, even postdifferentiated MEFs lacking p27KIP1 demonstrated a reduction in cell growth when passaged after BMP2 treatment, indicating that p27KIP1 contributes to (but is not solely responsible for) terminal growth arrest. We have not observed compensatory increases in p21CIP1 or p57KIP2 in p27KIP1- cultures (unpublished data), and the mechanisms accounting for the residual effects are not known.

Bottom Line: Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts.Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas.Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

View Article: PubMed Central - PubMed

Affiliation: Ian Potter Foundation Centre for Cancer Genomics and Predictive Medicine, and Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Center, Victoria, Melbourne, Australia. david.thomas@petermac.org

ABSTRACT
The molecular basis for the inverse relationship between differentiation and tumorigenesis is unknown. The function of runx2, a master regulator of osteoblast differentiation belonging to the runt family of tumor suppressor genes, is consistently disrupted in osteosarcoma cell lines. Ectopic expression of runx2 induces p27KIP1, thereby inhibiting the activity of S-phase cyclin complexes and leading to the dephosphorylation of the retinoblastoma tumor suppressor protein (pRb) and a G1 cell cycle arrest. Runx2 physically interacts with the hypophosphorylated form of pRb, a known coactivator of runx2, thereby completing a feed-forward loop in which progressive cell cycle exit promotes increased expression of the osteoblast phenotype. Loss of p27KIP1 perturbs transient and terminal cell cycle exit in osteoblasts. Consistent with the incompatibility of malignant transformation and permanent cell cycle exit, loss of p27KIP1 expression correlates with dedifferentiation in high-grade human osteosarcomas. Physiologic coupling of osteoblast differentiation to cell cycle withdrawal is mediated through runx2 and p27KIP1, and these processes are disrupted in osteosarcoma.

Show MeSH
Related in: MedlinePlus