Limits...
Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy.

Sanchez-Diaz PC, Burton TL, Burns SC, Hung JY, Penalva LO - BMC Cancer (2008)

Bottom Line: We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation.We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer.Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, TX, USA. sanchezdiaz@uthscsa.edu

ABSTRACT

Background: Musashi1 (Msi1) is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer.

Methods: We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells.

Results: We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.

Conclusion: Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

Show MeSH

Related in: MedlinePlus

Effect of Hedgehog blockade upon neurosphere formation. The ability to form neuropheres in the presence of the Hedgehog inhibitor cyclopamine was evaluated in Daoy cells. Spheres derived from Daoy control (upper) and Msi1 knockdown (KD; lower) were incubated for one week in the presence of inhibitor (Cyc) and tomatidine (Tom; negative control) at a concentration range of 5–20 μM. A reduction in neurosphere size was observed in both cell lines; however, the knockdown cell line appeared to be more sensitive to cyclopamine treatment as the effects upon sphere formation were significant (p < 0.05) at lower concentrations of inhibitor. a) Representative images of the effects upon neurosphere formation at 5 μM and 20 μM cyclopamine are shown. Scale bars represent 100 μm. b) The average number of neurospheres per field is represented for control and KD cells after treatment with tomatidine and cyclopamine. * indicates p < 0.05, ** p < 0.01 and *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2572071&req=5

Figure 5: Effect of Hedgehog blockade upon neurosphere formation. The ability to form neuropheres in the presence of the Hedgehog inhibitor cyclopamine was evaluated in Daoy cells. Spheres derived from Daoy control (upper) and Msi1 knockdown (KD; lower) were incubated for one week in the presence of inhibitor (Cyc) and tomatidine (Tom; negative control) at a concentration range of 5–20 μM. A reduction in neurosphere size was observed in both cell lines; however, the knockdown cell line appeared to be more sensitive to cyclopamine treatment as the effects upon sphere formation were significant (p < 0.05) at lower concentrations of inhibitor. a) Representative images of the effects upon neurosphere formation at 5 μM and 20 μM cyclopamine are shown. Scale bars represent 100 μm. b) The average number of neurospheres per field is represented for control and KD cells after treatment with tomatidine and cyclopamine. * indicates p < 0.05, ** p < 0.01 and *** p < 0.001.

Mentions: To determine the relevance of Hedgehog in cell proliferation in our model system, we performed a neurosphere assay in the presence of different concentrations of cyclopamine. Cyclopamine is a plant alkaloid which antagonizes Smoothened (Smo), a major signal transducer of the Hedgehog pathway [24]. As a negative control, we used the structural analog tomatidine at the same concentrations. While 5 μM cyclopamine induced a marked reduction in neurosphere size in the Msi1 knockdown cells (p < 0.05), 4 times higher concentrations were needed to elicit a significant effect in the control Daoy cells (p < 0.01; Figure 5). Therefore, Hedgehog seems to be important for neurosphere formation in Daoy cells, and the increased susceptibility to cyclopamine observed for Msi1 KD cells may reflect a partial down-regulation in the activity of this pathway.


Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy.

Sanchez-Diaz PC, Burton TL, Burns SC, Hung JY, Penalva LO - BMC Cancer (2008)

Effect of Hedgehog blockade upon neurosphere formation. The ability to form neuropheres in the presence of the Hedgehog inhibitor cyclopamine was evaluated in Daoy cells. Spheres derived from Daoy control (upper) and Msi1 knockdown (KD; lower) were incubated for one week in the presence of inhibitor (Cyc) and tomatidine (Tom; negative control) at a concentration range of 5–20 μM. A reduction in neurosphere size was observed in both cell lines; however, the knockdown cell line appeared to be more sensitive to cyclopamine treatment as the effects upon sphere formation were significant (p < 0.05) at lower concentrations of inhibitor. a) Representative images of the effects upon neurosphere formation at 5 μM and 20 μM cyclopamine are shown. Scale bars represent 100 μm. b) The average number of neurospheres per field is represented for control and KD cells after treatment with tomatidine and cyclopamine. * indicates p < 0.05, ** p < 0.01 and *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572071&req=5

Figure 5: Effect of Hedgehog blockade upon neurosphere formation. The ability to form neuropheres in the presence of the Hedgehog inhibitor cyclopamine was evaluated in Daoy cells. Spheres derived from Daoy control (upper) and Msi1 knockdown (KD; lower) were incubated for one week in the presence of inhibitor (Cyc) and tomatidine (Tom; negative control) at a concentration range of 5–20 μM. A reduction in neurosphere size was observed in both cell lines; however, the knockdown cell line appeared to be more sensitive to cyclopamine treatment as the effects upon sphere formation were significant (p < 0.05) at lower concentrations of inhibitor. a) Representative images of the effects upon neurosphere formation at 5 μM and 20 μM cyclopamine are shown. Scale bars represent 100 μm. b) The average number of neurospheres per field is represented for control and KD cells after treatment with tomatidine and cyclopamine. * indicates p < 0.05, ** p < 0.01 and *** p < 0.001.
Mentions: To determine the relevance of Hedgehog in cell proliferation in our model system, we performed a neurosphere assay in the presence of different concentrations of cyclopamine. Cyclopamine is a plant alkaloid which antagonizes Smoothened (Smo), a major signal transducer of the Hedgehog pathway [24]. As a negative control, we used the structural analog tomatidine at the same concentrations. While 5 μM cyclopamine induced a marked reduction in neurosphere size in the Msi1 knockdown cells (p < 0.05), 4 times higher concentrations were needed to elicit a significant effect in the control Daoy cells (p < 0.01; Figure 5). Therefore, Hedgehog seems to be important for neurosphere formation in Daoy cells, and the increased susceptibility to cyclopamine observed for Msi1 KD cells may reflect a partial down-regulation in the activity of this pathway.

Bottom Line: We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation.We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer.Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, TX, USA. sanchezdiaz@uthscsa.edu

ABSTRACT

Background: Musashi1 (Msi1) is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer.

Methods: We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells.

Results: We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy.

Conclusion: Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

Show MeSH
Related in: MedlinePlus