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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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Runx2-dependent osteogenic differentiation is disrupted in osteosarcoma cell lines. (A) Gene expression arrays were performed using RNA extracted from confluent cultures of SAOS2, MG63, HOS, B143, SJSA, and G292 cell lines, normalized to reference RNA as described in Materials and methods section. Data presented are the median log-transformed data for six cell lines. (B) Cells were transfected with 6ose2-luc (or with 6ose2mut-luc) and cytomegalo virus (CMV)–βgal plasmids. After 24 h, luciferase activity was measured and normalized to β-galactosidase. The ratio of the activity of 6ose2-luc to 6ose2mut-luc activity is shown. Ost, osteogenic osteoma cell line CCL-7672. Data shown are means ± SEM. (C) Cells were transfected with 6ose2-luc and CMV-βgal plasmids, with or without a runx2 expression vector. After 24 h, luciferase activity was measured and normalized for transfection efficiency with β-galactosidase. The ratio of luciferase activity in the presence or absence of runx2 is shown. Data shown are means ± SEM. (D) Western blot for runx2 and pRb in nuclear extracts from the indicated cell lines. HOb, human primary osteoblast.
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fig1: Runx2-dependent osteogenic differentiation is disrupted in osteosarcoma cell lines. (A) Gene expression arrays were performed using RNA extracted from confluent cultures of SAOS2, MG63, HOS, B143, SJSA, and G292 cell lines, normalized to reference RNA as described in Materials and methods section. Data presented are the median log-transformed data for six cell lines. (B) Cells were transfected with 6ose2-luc (or with 6ose2mut-luc) and cytomegalo virus (CMV)–βgal plasmids. After 24 h, luciferase activity was measured and normalized to β-galactosidase. The ratio of the activity of 6ose2-luc to 6ose2mut-luc activity is shown. Ost, osteogenic osteoma cell line CCL-7672. Data shown are means ± SEM. (C) Cells were transfected with 6ose2-luc and CMV-βgal plasmids, with or without a runx2 expression vector. After 24 h, luciferase activity was measured and normalized for transfection efficiency with β-galactosidase. The ratio of luciferase activity in the presence or absence of runx2 is shown. Data shown are means ± SEM. (D) Western blot for runx2 and pRb in nuclear extracts from the indicated cell lines. HOb, human primary osteoblast.

Mentions: We first used transcriptional profiling to objectively characterize the differentiation state of a panel of osteosarcoma cell lines (SAOS2, MG63, B143, HOS, SJSA, and G292) relative to an osteoblast-like reference. The reference consisted of primary stromal stem cells in which expression of markers of the mature osteoblast phenotype was induced by culture in the presence of ascorbic acid, dexamethasone, and inorganic phosphate (Gronthos et al., 2003). These markers include runx2, osterix, osteocalcin, and the ability to mineralize in vitro. Consistent with a transformed state, several putative oncogenes, including FOS and cyclins A1, B2, E1, and D1 (Fig. 1 A), were commonly overexpressed in osteosarcoma cell lines, whereas the Cdk inhibitors p16INK4A and p57KIP2 were relatively underexpressed. Shown in Fig. 1 A is the expression pattern of 16 bone-related genes selected from a previously published microarray study on the osteoblast phenotype (Balint et al., 2003). Twelve genes, including the key osteoblast transcription factor, RUNX2, were underexpressed across all osteosarcoma cell lines relative to our osteoblastic reference. Osteocalcin, a late and specific osteoblast marker not represented on our arrays, was undetectable by RT-PCR in the osteosarcoma lines (not depicted). Overall, the average median expression of 16 osteoblast-related genes in the osteosarcoma cell lines was reduced to 38 ± 8% of the osteoblastic reference.


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)

Runx2-dependent osteogenic differentiation is disrupted in osteosarcoma cell lines. (A) Gene expression arrays were performed using RNA extracted from confluent cultures of SAOS2, MG63, HOS, B143, SJSA, and G292 cell lines, normalized to reference RNA as described in Materials and methods section. Data presented are the median log-transformed data for six cell lines. (B) Cells were transfected with 6ose2-luc (or with 6ose2mut-luc) and cytomegalo virus (CMV)–βgal plasmids. After 24 h, luciferase activity was measured and normalized to β-galactosidase. The ratio of the activity of 6ose2-luc to 6ose2mut-luc activity is shown. Ost, osteogenic osteoma cell line CCL-7672. Data shown are means ± SEM. (C) Cells were transfected with 6ose2-luc and CMV-βgal plasmids, with or without a runx2 expression vector. After 24 h, luciferase activity was measured and normalized for transfection efficiency with β-galactosidase. The ratio of luciferase activity in the presence or absence of runx2 is shown. Data shown are means ± SEM. (D) Western blot for runx2 and pRb in nuclear extracts from the indicated cell lines. HOb, human primary osteoblast.
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Related In: Results  -  Collection

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fig1: Runx2-dependent osteogenic differentiation is disrupted in osteosarcoma cell lines. (A) Gene expression arrays were performed using RNA extracted from confluent cultures of SAOS2, MG63, HOS, B143, SJSA, and G292 cell lines, normalized to reference RNA as described in Materials and methods section. Data presented are the median log-transformed data for six cell lines. (B) Cells were transfected with 6ose2-luc (or with 6ose2mut-luc) and cytomegalo virus (CMV)–βgal plasmids. After 24 h, luciferase activity was measured and normalized to β-galactosidase. The ratio of the activity of 6ose2-luc to 6ose2mut-luc activity is shown. Ost, osteogenic osteoma cell line CCL-7672. Data shown are means ± SEM. (C) Cells were transfected with 6ose2-luc and CMV-βgal plasmids, with or without a runx2 expression vector. After 24 h, luciferase activity was measured and normalized for transfection efficiency with β-galactosidase. The ratio of luciferase activity in the presence or absence of runx2 is shown. Data shown are means ± SEM. (D) Western blot for runx2 and pRb in nuclear extracts from the indicated cell lines. HOb, human primary osteoblast.
Mentions: We first used transcriptional profiling to objectively characterize the differentiation state of a panel of osteosarcoma cell lines (SAOS2, MG63, B143, HOS, SJSA, and G292) relative to an osteoblast-like reference. The reference consisted of primary stromal stem cells in which expression of markers of the mature osteoblast phenotype was induced by culture in the presence of ascorbic acid, dexamethasone, and inorganic phosphate (Gronthos et al., 2003). These markers include runx2, osterix, osteocalcin, and the ability to mineralize in vitro. Consistent with a transformed state, several putative oncogenes, including FOS and cyclins A1, B2, E1, and D1 (Fig. 1 A), were commonly overexpressed in osteosarcoma cell lines, whereas the Cdk inhibitors p16INK4A and p57KIP2 were relatively underexpressed. Shown in Fig. 1 A is the expression pattern of 16 bone-related genes selected from a previously published microarray study on the osteoblast phenotype (Balint et al., 2003). Twelve genes, including the key osteoblast transcription factor, RUNX2, were underexpressed across all osteosarcoma cell lines relative to our osteoblastic reference. Osteocalcin, a late and specific osteoblast marker not represented on our arrays, was undetectable by RT-PCR in the osteosarcoma lines (not depicted). Overall, the average median expression of 16 osteoblast-related genes in the osteosarcoma cell lines was reduced to 38 ± 8% of the osteoblastic reference.

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