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Sonic hedgehog opposes epithelial cell cycle arrest.

Fan H, Khavari PA - J. Cell Biol. (1999)

Bottom Line: Shh-induced epidermal hyperplasia is accompanied by continued cell proliferation in normally growth arrested suprabasal cells in vivo.Shh-expressing cells fail to exit S and G2/M phases in response to calcium-induced differentiation and also resist exhaustion of replicative growth capacity.In addition, Shh blocks p21(CIP1/WAF1)-induced growth arrest.

View Article: PubMed Central - PubMed

Affiliation: VA Palo Alto Health Care System, Palo Alto, California 94304, USA.

ABSTRACT
Stratified epithelium displays an equilibrium between proliferation and cell cycle arrest, a balance that is disrupted in basal cell carcinoma (BCC). Sonic hedgehog (Shh) pathway activation appears sufficient to induce BCC, however, the way it does so is unknown. Shh-induced epidermal hyperplasia is accompanied by continued cell proliferation in normally growth arrested suprabasal cells in vivo. Shh-expressing cells fail to exit S and G2/M phases in response to calcium-induced differentiation and also resist exhaustion of replicative growth capacity. In addition, Shh blocks p21(CIP1/WAF1)-induced growth arrest. These data indicate that Shh promotes neoplasia by opposing normal stimuli for epithelial cell cycle arrest.

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Shh antagonizes p21Cip1-induced growth arrest. (a) Cell growth after p21Cip1 expression. 2 d after high efficiency gene transfer with Shh or GFP retroviral expression vectors, normal human epithelial cells were transduced in triplicate independent transductions at high efficiency with a retroviral expression vector for human p21Cip1 and cell numbers determined in the days following. Data is expressed from triplicate independent transductions ± SEM (b) SA-β-gal induction. Proportion of cells expressing SA-β-gal after p21Cip1 gene transfer. Arrows denote representative SA-β-gal[+] cells. Quantitation of percent SA-β-gal[+] cells ± SEM from three independent transductions is shown at right. (c) Shh effect on CDK2 and CDK4 kinase activity. Extracts from normal human epithelial cells transduced with retroviral expression vectors for Shh, GFP control as well as those cotransduced with vectors for either both Shh and p21Cip1 or GFP and p21Cip1 were immunoprecipitated using antibodies to CDK2 and CDK4. Kinase activity (top panels) was measured using histone H1 as a substrate. Immunoblotting was also performed in parallel to demonstrate the amount of CDK2 and CDK4 protein present in the precipitate (bottom panels below kinase activity).
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Figure 4: Shh antagonizes p21Cip1-induced growth arrest. (a) Cell growth after p21Cip1 expression. 2 d after high efficiency gene transfer with Shh or GFP retroviral expression vectors, normal human epithelial cells were transduced in triplicate independent transductions at high efficiency with a retroviral expression vector for human p21Cip1 and cell numbers determined in the days following. Data is expressed from triplicate independent transductions ± SEM (b) SA-β-gal induction. Proportion of cells expressing SA-β-gal after p21Cip1 gene transfer. Arrows denote representative SA-β-gal[+] cells. Quantitation of percent SA-β-gal[+] cells ± SEM from three independent transductions is shown at right. (c) Shh effect on CDK2 and CDK4 kinase activity. Extracts from normal human epithelial cells transduced with retroviral expression vectors for Shh, GFP control as well as those cotransduced with vectors for either both Shh and p21Cip1 or GFP and p21Cip1 were immunoprecipitated using antibodies to CDK2 and CDK4. Kinase activity (top panels) was measured using histone H1 as a substrate. Immunoblotting was also performed in parallel to demonstrate the amount of CDK2 and CDK4 protein present in the precipitate (bottom panels below kinase activity).

Mentions: Another negative growth regulatory mechanism bypassed in neoplasia are cyclin-dependent kinase inhibitors (CKIs). To determine if Shh could also confer resistance to CKI-induced growth arrest, we studied its impact on the effects of p21Cip1, a CKI of known importance in epithelial growth inhibition 823. p21Cip1 is known to be important in the cell growth arrest that occurs before keratinocyte terminal differentiation 823. Shh[+] and control cells were transduced at high efficiency with a retroviral expression vector for p21Cip1 and kinetics of cell growth were determined. As anticipated, p21Cip1 profoundly inhibits proliferation of both untransduced and GFP[+] control cells, however, p21Cip1 expression fails to inhibit exponential growth by Shh[+] cells (Fig. 4 a). In addition to resisting p21Cip1 growth arrest, Shh[+] cells fail to induce a biomarker seen in permanent cell cycle arrest, SA-β-gal 9, in response to p21Cip1. Whereas ∼50% of control cells are SA-β-gal[+] by day 5, only 15% of Shh[+] cells express this biomarker (Fig. 4 b). Shh expression in these cells is also associated with augmented levels of CDK2 and CDK4 associated kinase activity, with this activity not fully repressed by p21Cip1 in the case of CDK2 (Fig. 4 c). These data indicate that Shh opposes p21Cip1-induced growth arrest and suggest that Shh leads to activation of key core cell cycle machinery elements CDK2 and CDK4.


Sonic hedgehog opposes epithelial cell cycle arrest.

Fan H, Khavari PA - J. Cell Biol. (1999)

Shh antagonizes p21Cip1-induced growth arrest. (a) Cell growth after p21Cip1 expression. 2 d after high efficiency gene transfer with Shh or GFP retroviral expression vectors, normal human epithelial cells were transduced in triplicate independent transductions at high efficiency with a retroviral expression vector for human p21Cip1 and cell numbers determined in the days following. Data is expressed from triplicate independent transductions ± SEM (b) SA-β-gal induction. Proportion of cells expressing SA-β-gal after p21Cip1 gene transfer. Arrows denote representative SA-β-gal[+] cells. Quantitation of percent SA-β-gal[+] cells ± SEM from three independent transductions is shown at right. (c) Shh effect on CDK2 and CDK4 kinase activity. Extracts from normal human epithelial cells transduced with retroviral expression vectors for Shh, GFP control as well as those cotransduced with vectors for either both Shh and p21Cip1 or GFP and p21Cip1 were immunoprecipitated using antibodies to CDK2 and CDK4. Kinase activity (top panels) was measured using histone H1 as a substrate. Immunoblotting was also performed in parallel to demonstrate the amount of CDK2 and CDK4 protein present in the precipitate (bottom panels below kinase activity).
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Related In: Results  -  Collection

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Figure 4: Shh antagonizes p21Cip1-induced growth arrest. (a) Cell growth after p21Cip1 expression. 2 d after high efficiency gene transfer with Shh or GFP retroviral expression vectors, normal human epithelial cells were transduced in triplicate independent transductions at high efficiency with a retroviral expression vector for human p21Cip1 and cell numbers determined in the days following. Data is expressed from triplicate independent transductions ± SEM (b) SA-β-gal induction. Proportion of cells expressing SA-β-gal after p21Cip1 gene transfer. Arrows denote representative SA-β-gal[+] cells. Quantitation of percent SA-β-gal[+] cells ± SEM from three independent transductions is shown at right. (c) Shh effect on CDK2 and CDK4 kinase activity. Extracts from normal human epithelial cells transduced with retroviral expression vectors for Shh, GFP control as well as those cotransduced with vectors for either both Shh and p21Cip1 or GFP and p21Cip1 were immunoprecipitated using antibodies to CDK2 and CDK4. Kinase activity (top panels) was measured using histone H1 as a substrate. Immunoblotting was also performed in parallel to demonstrate the amount of CDK2 and CDK4 protein present in the precipitate (bottom panels below kinase activity).
Mentions: Another negative growth regulatory mechanism bypassed in neoplasia are cyclin-dependent kinase inhibitors (CKIs). To determine if Shh could also confer resistance to CKI-induced growth arrest, we studied its impact on the effects of p21Cip1, a CKI of known importance in epithelial growth inhibition 823. p21Cip1 is known to be important in the cell growth arrest that occurs before keratinocyte terminal differentiation 823. Shh[+] and control cells were transduced at high efficiency with a retroviral expression vector for p21Cip1 and kinetics of cell growth were determined. As anticipated, p21Cip1 profoundly inhibits proliferation of both untransduced and GFP[+] control cells, however, p21Cip1 expression fails to inhibit exponential growth by Shh[+] cells (Fig. 4 a). In addition to resisting p21Cip1 growth arrest, Shh[+] cells fail to induce a biomarker seen in permanent cell cycle arrest, SA-β-gal 9, in response to p21Cip1. Whereas ∼50% of control cells are SA-β-gal[+] by day 5, only 15% of Shh[+] cells express this biomarker (Fig. 4 b). Shh expression in these cells is also associated with augmented levels of CDK2 and CDK4 associated kinase activity, with this activity not fully repressed by p21Cip1 in the case of CDK2 (Fig. 4 c). These data indicate that Shh opposes p21Cip1-induced growth arrest and suggest that Shh leads to activation of key core cell cycle machinery elements CDK2 and CDK4.

Bottom Line: Shh-induced epidermal hyperplasia is accompanied by continued cell proliferation in normally growth arrested suprabasal cells in vivo.Shh-expressing cells fail to exit S and G2/M phases in response to calcium-induced differentiation and also resist exhaustion of replicative growth capacity.In addition, Shh blocks p21(CIP1/WAF1)-induced growth arrest.

View Article: PubMed Central - PubMed

Affiliation: VA Palo Alto Health Care System, Palo Alto, California 94304, USA.

ABSTRACT
Stratified epithelium displays an equilibrium between proliferation and cell cycle arrest, a balance that is disrupted in basal cell carcinoma (BCC). Sonic hedgehog (Shh) pathway activation appears sufficient to induce BCC, however, the way it does so is unknown. Shh-induced epidermal hyperplasia is accompanied by continued cell proliferation in normally growth arrested suprabasal cells in vivo. Shh-expressing cells fail to exit S and G2/M phases in response to calcium-induced differentiation and also resist exhaustion of replicative growth capacity. In addition, Shh blocks p21(CIP1/WAF1)-induced growth arrest. These data indicate that Shh promotes neoplasia by opposing normal stimuli for epithelial cell cycle arrest.

Show MeSH
Related in: MedlinePlus