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Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGF.

Podergajs N, Brekka N, Radlwimmer B, Herold-Mende C, Talasila KM, Tiemann K, Rajcevic U, Lah TT, Bjerkvig R, Miletic H - Radiol Oncol (2013)

Bottom Line: In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF.Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect.We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia.

ABSTRACT

Background: Patient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stem-like cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF.

Materials and methods: [corrected] To address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification.

Results: Although both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).

Conclusions: We demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.

No MeSH data available.


Related in: MedlinePlus

bFGF stimulates growth of GSCs due to increased viability or proliferation. (A) Cell cycle analysis of cells grown in the presence of EGF or bFGF. The relative changes in the cell number in G1, S and G2 phase were measured as indicators of cell proliferation. bFGF increased the number of cells in S phase in NCH421k cells, while there were no effects on the cell cycle in NCH644 cells. (B) Apoptosis assay of cells double labeled with Annexin V-FITC and PI. Percentages of cells in each histogram are representative of early apoptosis (labeled Annexin V), necrosis (labeled PI) and late apoptosis (labeled Annexin V+PI). Number of apoptotic NCH644 cells was decreased in presence of bFGF, but not in the presence of EGF. No significant changes upon EGF or bFGF stimulation were observed in the NCH421k cell line compared to the medium without growth factors. Labels: NB= Neurobasal medium without growth factors; compl=NB with EGF and bFGF; EGF=NB with EGF; bFGF=NB with bFGF. Data shown are mean ± SD of three independent experiments. **: p<0,01.
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f4-rado-47-04-330: bFGF stimulates growth of GSCs due to increased viability or proliferation. (A) Cell cycle analysis of cells grown in the presence of EGF or bFGF. The relative changes in the cell number in G1, S and G2 phase were measured as indicators of cell proliferation. bFGF increased the number of cells in S phase in NCH421k cells, while there were no effects on the cell cycle in NCH644 cells. (B) Apoptosis assay of cells double labeled with Annexin V-FITC and PI. Percentages of cells in each histogram are representative of early apoptosis (labeled Annexin V), necrosis (labeled PI) and late apoptosis (labeled Annexin V+PI). Number of apoptotic NCH644 cells was decreased in presence of bFGF, but not in the presence of EGF. No significant changes upon EGF or bFGF stimulation were observed in the NCH421k cell line compared to the medium without growth factors. Labels: NB= Neurobasal medium without growth factors; compl=NB with EGF and bFGF; EGF=NB with EGF; bFGF=NB with bFGF. Data shown are mean ± SD of three independent experiments. **: p<0,01.

Mentions: We then determined how bFGF and EGF affect cell cycle parameters and apoptosis. Cells were stimulated for 48 hours with bFGF or EGF. We observed that NCH421k cells showed a significant (p<0.01) increase in cells entering the S phase upon bFGF stimulation, verifying that the growth of NCH421k cells is stimulated by bFGF (Figure 4A). Stimulation with EGF had no effect on the cell cycle in NCH421k. Surprisingly, NCH644 cells did not show a significant difference upon stimulation with bFGF or EGF. Therefore, we used a cell death assay to analyze whether there is a difference in apoptosis. We observed 1.1–fold (p<0.01) less apoptotic NCH644 cells upon bFGF stimulation compared to unstimulated cells. EGF stimulation did not have this effect. In contrast to NCH644, the NCH421k cell line did not show any significant differences in the apoptosis assay (Figure 4B). Thus, in both GSC lines bFGF stimulated growth by either enhancing proliferation (NCH421k) or mediating resistance to apoptosis (NCH644), while EGF showed no effects.


Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGF.

Podergajs N, Brekka N, Radlwimmer B, Herold-Mende C, Talasila KM, Tiemann K, Rajcevic U, Lah TT, Bjerkvig R, Miletic H - Radiol Oncol (2013)

bFGF stimulates growth of GSCs due to increased viability or proliferation. (A) Cell cycle analysis of cells grown in the presence of EGF or bFGF. The relative changes in the cell number in G1, S and G2 phase were measured as indicators of cell proliferation. bFGF increased the number of cells in S phase in NCH421k cells, while there were no effects on the cell cycle in NCH644 cells. (B) Apoptosis assay of cells double labeled with Annexin V-FITC and PI. Percentages of cells in each histogram are representative of early apoptosis (labeled Annexin V), necrosis (labeled PI) and late apoptosis (labeled Annexin V+PI). Number of apoptotic NCH644 cells was decreased in presence of bFGF, but not in the presence of EGF. No significant changes upon EGF or bFGF stimulation were observed in the NCH421k cell line compared to the medium without growth factors. Labels: NB= Neurobasal medium without growth factors; compl=NB with EGF and bFGF; EGF=NB with EGF; bFGF=NB with bFGF. Data shown are mean ± SD of three independent experiments. **: p<0,01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3814277&req=5

f4-rado-47-04-330: bFGF stimulates growth of GSCs due to increased viability or proliferation. (A) Cell cycle analysis of cells grown in the presence of EGF or bFGF. The relative changes in the cell number in G1, S and G2 phase were measured as indicators of cell proliferation. bFGF increased the number of cells in S phase in NCH421k cells, while there were no effects on the cell cycle in NCH644 cells. (B) Apoptosis assay of cells double labeled with Annexin V-FITC and PI. Percentages of cells in each histogram are representative of early apoptosis (labeled Annexin V), necrosis (labeled PI) and late apoptosis (labeled Annexin V+PI). Number of apoptotic NCH644 cells was decreased in presence of bFGF, but not in the presence of EGF. No significant changes upon EGF or bFGF stimulation were observed in the NCH421k cell line compared to the medium without growth factors. Labels: NB= Neurobasal medium without growth factors; compl=NB with EGF and bFGF; EGF=NB with EGF; bFGF=NB with bFGF. Data shown are mean ± SD of three independent experiments. **: p<0,01.
Mentions: We then determined how bFGF and EGF affect cell cycle parameters and apoptosis. Cells were stimulated for 48 hours with bFGF or EGF. We observed that NCH421k cells showed a significant (p<0.01) increase in cells entering the S phase upon bFGF stimulation, verifying that the growth of NCH421k cells is stimulated by bFGF (Figure 4A). Stimulation with EGF had no effect on the cell cycle in NCH421k. Surprisingly, NCH644 cells did not show a significant difference upon stimulation with bFGF or EGF. Therefore, we used a cell death assay to analyze whether there is a difference in apoptosis. We observed 1.1–fold (p<0.01) less apoptotic NCH644 cells upon bFGF stimulation compared to unstimulated cells. EGF stimulation did not have this effect. In contrast to NCH644, the NCH421k cell line did not show any significant differences in the apoptosis assay (Figure 4B). Thus, in both GSC lines bFGF stimulated growth by either enhancing proliferation (NCH421k) or mediating resistance to apoptosis (NCH644), while EGF showed no effects.

Bottom Line: In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF.Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect.We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia.

ABSTRACT

Background: Patient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stem-like cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF.

Materials and methods: [corrected] To address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification.

Results: Although both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).

Conclusions: We demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.

No MeSH data available.


Related in: MedlinePlus