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A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.

Cong F, Zhang J, Pao W, Zhou P, Varmus H - BMC Mol. Biol. (2003)

Bottom Line: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate.As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. congf@mskcc.org

ABSTRACT

Background: The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic beta-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either beta-catenin or adenomatous polyposis coli (APC), renders beta-catenin resistant to degradation, and has been associated with multiple types of human cancers.

Results: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the beta-catenin binding domain of E-cadherin fused to betaTrCP ubiquitin-protein ligase, the stable beta-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type beta-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of betaTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of beta-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.

Conclusion: We have designed a novel approach to induce degradation of stabilized/mutated beta-catenin. Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment.

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F-TrCP-Ecad inhibits clonogenic and tumorigenic potential of DLD1 cells. A. βTrCP-Ecad inhibits the growth rate of DLD1 cells. DLD1-F-TrCP-Ecad cells were grown in the presence or the absence of Dox for five days. The numbers of cells were counted daily in triplicates. B. Induction of F-TrCP-Ecad decreases the colony forming ability of DLD1 cells. DLD1-F-TrCP-Ecad cells were diluted, plated at a low density, and cultured in the presence or the absence of Dox for 14 days. Plates were photographed (top panel) and the number of colonies was counted (bottom panel). The experiment was performed in triplicates C. F-TrCP-Ecad induction inhibits tumorigenicity of DLD1 cells in nude mice. 2 × 106 DLD1-F-TrCP-Ecad cells were injected subcutaneously in nude mice. Each mouse received three injections. Nude mice were fed with either regular or Dox-containing food, and photographed three weeks after injection. In this observation period, six of six injections gave rise to tumors (indicated with asterisks) on mice fed with Dox-impregnated food, while none of the six injections generated tumor on mice fed with regular food.
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Figure 4: F-TrCP-Ecad inhibits clonogenic and tumorigenic potential of DLD1 cells. A. βTrCP-Ecad inhibits the growth rate of DLD1 cells. DLD1-F-TrCP-Ecad cells were grown in the presence or the absence of Dox for five days. The numbers of cells were counted daily in triplicates. B. Induction of F-TrCP-Ecad decreases the colony forming ability of DLD1 cells. DLD1-F-TrCP-Ecad cells were diluted, plated at a low density, and cultured in the presence or the absence of Dox for 14 days. Plates were photographed (top panel) and the number of colonies was counted (bottom panel). The experiment was performed in triplicates C. F-TrCP-Ecad induction inhibits tumorigenicity of DLD1 cells in nude mice. 2 × 106 DLD1-F-TrCP-Ecad cells were injected subcutaneously in nude mice. Each mouse received three injections. Nude mice were fed with either regular or Dox-containing food, and photographed three weeks after injection. In this observation period, six of six injections gave rise to tumors (indicated with asterisks) on mice fed with Dox-impregnated food, while none of the six injections generated tumor on mice fed with regular food.

Mentions: To study the effect of F-TrCP-Ecad induction on cell proliferation, we first measured the growth rate of DLD1-F-TrCP-Ecad cells in the presence or absence of Dox. A reduced rate of cell growth was observed when cells were cultured in the absence of Dox (Fig. 4A). This effect of F-TrCP-Ecad on cell proliferation was not a result of increased cell death, as determined by trypan blue staining (data not shown). A clonogenic assay was performed, in which DLD1-F-TrCP-Ecad cells were plated at a low density (2,000 cells/10 cm plate) and cultured in the presence or absence of Dox. Induction of F-TrCP-Ecad expression dramatically reduced the colony forming capability of DLD1 cells (Fig. 4B).


A protein knockdown strategy to study the function of beta-catenin in tumorigenesis.

Cong F, Zhang J, Pao W, Zhou P, Varmus H - BMC Mol. Biol. (2003)

F-TrCP-Ecad inhibits clonogenic and tumorigenic potential of DLD1 cells. A. βTrCP-Ecad inhibits the growth rate of DLD1 cells. DLD1-F-TrCP-Ecad cells were grown in the presence or the absence of Dox for five days. The numbers of cells were counted daily in triplicates. B. Induction of F-TrCP-Ecad decreases the colony forming ability of DLD1 cells. DLD1-F-TrCP-Ecad cells were diluted, plated at a low density, and cultured in the presence or the absence of Dox for 14 days. Plates were photographed (top panel) and the number of colonies was counted (bottom panel). The experiment was performed in triplicates C. F-TrCP-Ecad induction inhibits tumorigenicity of DLD1 cells in nude mice. 2 × 106 DLD1-F-TrCP-Ecad cells were injected subcutaneously in nude mice. Each mouse received three injections. Nude mice were fed with either regular or Dox-containing food, and photographed three weeks after injection. In this observation period, six of six injections gave rise to tumors (indicated with asterisks) on mice fed with Dox-impregnated food, while none of the six injections generated tumor on mice fed with regular food.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC222962&req=5

Figure 4: F-TrCP-Ecad inhibits clonogenic and tumorigenic potential of DLD1 cells. A. βTrCP-Ecad inhibits the growth rate of DLD1 cells. DLD1-F-TrCP-Ecad cells were grown in the presence or the absence of Dox for five days. The numbers of cells were counted daily in triplicates. B. Induction of F-TrCP-Ecad decreases the colony forming ability of DLD1 cells. DLD1-F-TrCP-Ecad cells were diluted, plated at a low density, and cultured in the presence or the absence of Dox for 14 days. Plates were photographed (top panel) and the number of colonies was counted (bottom panel). The experiment was performed in triplicates C. F-TrCP-Ecad induction inhibits tumorigenicity of DLD1 cells in nude mice. 2 × 106 DLD1-F-TrCP-Ecad cells were injected subcutaneously in nude mice. Each mouse received three injections. Nude mice were fed with either regular or Dox-containing food, and photographed three weeks after injection. In this observation period, six of six injections gave rise to tumors (indicated with asterisks) on mice fed with Dox-impregnated food, while none of the six injections generated tumor on mice fed with regular food.
Mentions: To study the effect of F-TrCP-Ecad induction on cell proliferation, we first measured the growth rate of DLD1-F-TrCP-Ecad cells in the presence or absence of Dox. A reduced rate of cell growth was observed when cells were cultured in the absence of Dox (Fig. 4A). This effect of F-TrCP-Ecad on cell proliferation was not a result of increased cell death, as determined by trypan blue staining (data not shown). A clonogenic assay was performed, in which DLD1-F-TrCP-Ecad cells were plated at a low density (2,000 cells/10 cm plate) and cultured in the presence or absence of Dox. Induction of F-TrCP-Ecad expression dramatically reduced the colony forming capability of DLD1 cells (Fig. 4B).

Bottom Line: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate.As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. congf@mskcc.org

ABSTRACT

Background: The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic beta-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either beta-catenin or adenomatous polyposis coli (APC), renders beta-catenin resistant to degradation, and has been associated with multiple types of human cancers.

Results: A protein knockdown strategy was designed to reduce the cytosolic beta-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the beta-catenin binding domain of E-cadherin fused to betaTrCP ubiquitin-protein ligase, the stable beta-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor) ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type beta-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of betaTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of beta-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice.

Conclusion: We have designed a novel approach to induce degradation of stabilized/mutated beta-catenin. Our results suggest that a high concentration of cytoplasmic beta-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment.

Show MeSH
Related in: MedlinePlus