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

Neurosphere formation assay. a) Second generation of neurospheres derived from Daoy control (upper) and knockdown (KD; lower) at two different cell densities (500 and 250 cells/well) are shown. The smaller size neurospheres detected in the knockdown suggested that Msi1 controls cell proliferation in Daoy. b) The average number of spheres per well is represented at different cell densities (500, 250, 125 and 75 cells/well). Only spheres larger than 50 μm in diameter were scored. c) The differences in neurosphere size are represented at different cell densities (500, 250, 125 and 75 cells/well). * indicates p < 0.05 and ** p < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Neurosphere formation assay. a) Second generation of neurospheres derived from Daoy control (upper) and knockdown (KD; lower) at two different cell densities (500 and 250 cells/well) are shown. The smaller size neurospheres detected in the knockdown suggested that Msi1 controls cell proliferation in Daoy. b) The average number of spheres per well is represented at different cell densities (500, 250, 125 and 75 cells/well). Only spheres larger than 50 μm in diameter were scored. c) The differences in neurosphere size are represented at different cell densities (500, 250, 125 and 75 cells/well). * indicates p < 0.05 and ** p < 0.0001.

Mentions: Msi1 is a stem cell marker reported to be essential for maintenance of stem cells [1,3,4]. Cells with stem-like properties have been isolated in many human brain cancers including medulloblastoma (reviewed in [22]). Since high levels of Msi1 are frequently found in medulloblastoma and since we observed that Msi1 expression was elevated in Daoy neurosphere compared to monolayer cultures (Figure 1), we asked if Msi1 might be involved in regulating proliferation in cancer cells. Here we evaluated neurosphere formation as an indicator of cell proliferative potential. Single-cell suspensions were plated at clonal density to minimize the effects of cell aggregation in favor of single-cell sphere generation. After 10 days of incubation, neurospheres larger than 50 μm in diameter were counted. As shown in Figure 3, a significant reduction in both number and overall size of the spheres was detected when Msi1 expression was depleted. The defect observed in sphere formation was more severe in subsequent serial passages (data not shown), thus indicating a role for Msi1 in sustaining cancer cells.


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)

Neurosphere formation assay. a) Second generation of neurospheres derived from Daoy control (upper) and knockdown (KD; lower) at two different cell densities (500 and 250 cells/well) are shown. The smaller size neurospheres detected in the knockdown suggested that Msi1 controls cell proliferation in Daoy. b) The average number of spheres per well is represented at different cell densities (500, 250, 125 and 75 cells/well). Only spheres larger than 50 μm in diameter were scored. c) The differences in neurosphere size are represented at different cell densities (500, 250, 125 and 75 cells/well). * indicates p < 0.05 and ** p < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Neurosphere formation assay. a) Second generation of neurospheres derived from Daoy control (upper) and knockdown (KD; lower) at two different cell densities (500 and 250 cells/well) are shown. The smaller size neurospheres detected in the knockdown suggested that Msi1 controls cell proliferation in Daoy. b) The average number of spheres per well is represented at different cell densities (500, 250, 125 and 75 cells/well). Only spheres larger than 50 μm in diameter were scored. c) The differences in neurosphere size are represented at different cell densities (500, 250, 125 and 75 cells/well). * indicates p < 0.05 and ** p < 0.0001.
Mentions: Msi1 is a stem cell marker reported to be essential for maintenance of stem cells [1,3,4]. Cells with stem-like properties have been isolated in many human brain cancers including medulloblastoma (reviewed in [22]). Since high levels of Msi1 are frequently found in medulloblastoma and since we observed that Msi1 expression was elevated in Daoy neurosphere compared to monolayer cultures (Figure 1), we asked if Msi1 might be involved in regulating proliferation in cancer cells. Here we evaluated neurosphere formation as an indicator of cell proliferative potential. Single-cell suspensions were plated at clonal density to minimize the effects of cell aggregation in favor of single-cell sphere generation. After 10 days of incubation, neurospheres larger than 50 μm in diameter were counted. As shown in Figure 3, a significant reduction in both number and overall size of the spheres was detected when Msi1 expression was depleted. The defect observed in sphere formation was more severe in subsequent serial passages (data not shown), thus indicating a role for Msi1 in sustaining cancer cells.

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