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Published on: March 28, 2021
Temozolomide promotes genomic and phenotypic changes in glioblastoma cells
Aleksei A Stepanenko1, Svitlana V Andreieva1, Kateryna V Korets1
1Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Zabolotnogo str. 150, Kiev, 03680 Ukraine.
Background:
Temozolomide (TMZ) is a first-line drug for the treatment of glioblastoma. Long-term TMZ-treated tumour cells acquire TMZ resistance by profound reprogramming of the transcriptome, proteome, kinome, metabolism, and demonstrate versatile and opposite changes in proliferation, invasion, in vivo growth, and drug cross-resistance. We hypothesized that chromosomal instability (CIN) may be implicated in the generation of TMZ-driven molecular and phenotype diversity. CIN refers to the rate (cell-to-cell variability) with which whole chromosomes or portions of chromosomes are gained or lost.
Methods:
The long-term TMZ-treated cell lines were established in vitro (U251TMZ1, U251TMZ2, T98GTMZ and C6TMZ) and in vivo (C6R2TMZ). A glioma model was achieved by the intracerebral stereotactic implantation of C6 cells into the striatum region of rats. Genomic and phenotypic changes were analyzed by conventional cytogenetics, array CGH, trypan blue exclusion assay, soft agar colony formation assay, scratch wound healing assay, transwell invasion assay, quantitative polymerase chain reaction, and Western blotting.
Results:
Long-term TMZ treatment increased CIN-mediated genomic diversity in U251TMZ1, U251TMZ2 and T98GTMZ cells but reduced it in C6TMZ and C6R2TMZ cells. U251TMZ1 and U251TMZ2 cell lines, established in parallel with a similar treatment procedure with the only difference in the duration of treatment, underwent individual phenotypic changes. U251TMZ1 had a reduced proliferation and invasion but increased migration, whereas U251TMZ2 had an enhanced proliferation and invasion but no changes in migration. U251TMZ1 and U251TMZ2 cells demonstrated individual patterns in expression/activation of signal transduction proteins (e.g., MDM2, p53, ERK, AKT, and ASK). C6TMZ and C6R2TMZ cells had lower proliferation, colony formation efficiency and migration, whereas T98GTMZ cells had increased colony formation efficiency without any changes in proliferation, migration, and invasion. TMZ-treated lines demonstrated a differential response to a reduction in glucose concentration and an increased resistance to TMZ re-challenge but not temsirolimus (mTOR inhibitor) or U0126 (MEK1/2 inhibitor) treatment.
Conclusion:
Long-term TMZ treatment selected resistant genotype-phenotype variants or generated novel versatile phenotypes by increasing CIN. An increase of resistance to TMZ re-challenge seems to be the only predictable trait intrinsic to all long-term TMZ-treated tumour cells. Changes in genomic diversity may be responsible for heterogeneous phenotypes of TMZ-treated cell lines.
Insights
Long-term temozolomide treatment in glioblastoma cells can lead to drug resistance and diverse cellular changes. Chromosomal instability (CIN) plays a role in generating these varied responses and resistance patterns.
Area of Science:
- Oncology
- Genetics
- Cell Biology
Background:
- Temozolomide (TMZ) is a primary treatment for glioblastoma.
- Long-term TMZ treatment induces resistance through extensive molecular reprogramming.
- Chromosomal instability (CIN) is hypothesized to drive TMZ-induced diversity.
Purpose of the Study:
- To investigate the role of chromosomal instability (CIN) in generating molecular and phenotypic diversity in long-term temozolomide (TMZ)-treated glioblastoma cells.
- To analyze the genomic and phenotypic changes associated with acquired TMZ resistance.
- To understand the mechanisms underlying differential responses to TMZ treatment.
Main Methods:
- Established long-term TMZ-treated glioblastoma cell lines in vitro and in vivo.
- Utilized conventional cytogenetics, array comparative genomic hybridization (aCGH), and molecular assays (qPCR, Western blotting).
- Assessed phenotypic changes including proliferation, invasion, migration, and colony formation.
Main Results:
- Long-term TMZ treatment differentially affected CIN levels, increasing it in some lines and decreasing it in others.
- TMZ-resistant cell lines exhibited diverse phenotypic changes, including variations in proliferation, invasion, and migration.
- Acquired resistance to TMZ re-challenge was observed across treated cell lines, independent of specific phenotypic alterations.
Conclusions:
- Long-term TMZ treatment selects for or generates resistant variants through increased CIN.
- Acquired resistance to subsequent TMZ treatment is a consistent outcome of long-term exposure.
- Genomic diversity driven by CIN contributes to the heterogeneous phenotypes observed in TMZ-treated glioblastoma cells.
