Phenotypical Flexibility of the EGFRvIII-Positive Glioblastoma Cell Line and the Multidirectional Influence of TGFβ and EGF on These Cells—EGFRvIII Appears as a Weak Oncogene
Abstract
:1. Introduction
2. Results
2.1. DK-MG Subline Characterization
2.2. EGF Influence on DK-MG Sublines
2.3. TGFβ Influence on DK-MG Sublines
2.4. Characteristics and Influence of Analyzed Growth Factors on Primary EGFRvIII-Positive Glioblastomas
3. Discussion
4. Materials and Methods
4.1. Cell Cultures
4.2. Development of Cell Lines with Exogenous EGFRvIII and K-RASG12V Expression
4.3. Real-Time PCR
4.4. EGFR Total DNA Copy Number Calculation
4.5. Multiplex Ligation-Dependent Probe Amplification (MLPA)
4.6. Proliferation and Apoptosis Assessment
4.7. Western Blotting
4.8. Immunocytochemical Staining
4.9. Senescence Associated (SA)-β-Galactosidase Assay
4.10. Bromodeoxyuridine (BrdU) Incorporation Assay
4.11. Next-Generation Sequencing (NGS)
4.12. IC50 Determination
4.13. Statistical Analyses
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Cell Line | Gene ID | Type | Frequency % | Allele Call | Chromosome | Position | Reference | Variant | Coverage |
---|---|---|---|---|---|---|---|---|---|
Only DKMG low | CSF1R | MNP | 99.1 | Homozygous | 5 | 149,433,596 | TG | GA | 106 |
Only DKMG low | APC | SNP | 50.6 | Heterozygous | 5 | 112,175,770 | G | A | 915 |
DKMG low and high | KDR | SNP | 100 | Homozygous | 4 | 55,980,239 | C | T | 495 |
DKMG low and high | RET | SNP | 100 | Homozygous | 10 | 43,613,843 | G | T | 253 |
DKMG low and high | FLT3 | SNP | 100 | Homozygous | 13 | 28,610,183 | A | G | 459 |
DKMG low and high | TP53 | SNP | 52.8 | Heterozygous | 17 | 7,579,472 | G | C | 303 |
Cell Subline | EGFR Total DNA Copy Number | EGFR WT cDNA Relative Expression | EGFR vIII cDNA Relative Expression | % EGFR vIII Positive Cells | Doubling Time |
---|---|---|---|---|---|
DK-MGlow | 25 | 4.05 | 0.1 | 1–5% | 44 h |
DK-MGhigh | 218 | 4.25 | 371 | 40–60% | 23 h |
DK-MGextra-high | 196 | 3.80 | 289 | 100% | 14 h |
DK-MGlow exo vIII | 223 | 4.46 | 325 | 100% | 43 h |
DK-MGhigh exo vIII | 226 | 4.53 | 382 | 100% | 23 h |
Cell Line | DK-MG High | NIH/3T3 | Pericytes | Neural Stem Cells | Primary GB1 | Primary GB4 | Primary GB7 |
---|---|---|---|---|---|---|---|
IC50 value concentration range | 4 ng/mL | 1000 ng/mL | >1000 ng/mL | >1000 ng/mL | 14 ng/mL | >1000 ng/mL | >1000 ng/mL |
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Włodarczyk, A.; Tręda, C.; Rutkowska, A.; Grot, D.; Dobrewa, W.; Kierasińska, A.; Węgierska, M.; Wasiak, T.; Strózik, T.; Rieske, P.; et al. Phenotypical Flexibility of the EGFRvIII-Positive Glioblastoma Cell Line and the Multidirectional Influence of TGFβ and EGF on These Cells—EGFRvIII Appears as a Weak Oncogene. Int. J. Mol. Sci. 2022, 23, 12129. https://doi.org/10.3390/ijms232012129
Włodarczyk A, Tręda C, Rutkowska A, Grot D, Dobrewa W, Kierasińska A, Węgierska M, Wasiak T, Strózik T, Rieske P, et al. Phenotypical Flexibility of the EGFRvIII-Positive Glioblastoma Cell Line and the Multidirectional Influence of TGFβ and EGF on These Cells—EGFRvIII Appears as a Weak Oncogene. International Journal of Molecular Sciences. 2022; 23(20):12129. https://doi.org/10.3390/ijms232012129
Chicago/Turabian StyleWłodarczyk, Aneta, Cezary Tręda, Adrianna Rutkowska, Dagmara Grot, Weronika Dobrewa, Amelia Kierasińska, Marta Węgierska, Tomasz Wasiak, Tadeusz Strózik, Piotr Rieske, and et al. 2022. "Phenotypical Flexibility of the EGFRvIII-Positive Glioblastoma Cell Line and the Multidirectional Influence of TGFβ and EGF on These Cells—EGFRvIII Appears as a Weak Oncogene" International Journal of Molecular Sciences 23, no. 20: 12129. https://doi.org/10.3390/ijms232012129