title: Temozolomide promotes genomic and phenotypic changes in glioblastoma cells.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Zabolotnogo str. 150, Kiev, 03680 Ukraine.
authors.forename: Aleksei A
authors.initials: AA
authors.lastname: Stepanenko
authors.affiliation: Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Zabolotnogo str. 150, Kiev, 03680 Ukraine.
authors.forename: Svitlana V
authors.initials: SV
authors.lastname: Andreieva
authors.affiliation: Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Zabolotnogo str. 150, Kiev, 03680 Ukraine.
authors.forename: Kateryna V
authors.initials: KV
authors.lastname: Korets
authors.affiliation: Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Zabolotnogo str. 150, Kiev, 03680 Ukraine.
authors.forename: Dmytro O
authors.initials: DO
authors.lastname: Mykytenko
authors.affiliation: Department of Medicinal Nanobiotechnology, Pirogov Russian State Medical University, Ostrovitianov str. 1, Moscow, 117997 Russia ; Federal Research and Clinical Centre, FMBA of Russia, Orekhoviy Bulvar str. 28, Moscow, 115682 Russia.
authors.forename: Vladimir P
authors.initials: VP
authors.lastname: Baklaushev
authors.affiliation: Department of Diagnostic of Hereditary Pathology, Institute of Hereditary Pathology, National Academy of Medical Sciences of Ukraine, Lysenko str. 31A, Lviv, 79008 Ukraine.
authors.forename: Nataliya L
authors.initials: NL
authors.lastname: Huleyuk
authors.affiliation: Department of Experimental Cell System, R.E.Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Science of Ukraine, Vasylkivska str. 45, Kiev, 03022 Ukraine.
authors.forename: Oksana A
authors.initials: OA
authors.lastname: Kovalova
authors.affiliation: Department of Human Genetics, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Zabolotnogo str. 150, Kiev, 03680 Ukraine.
authors.forename: Kateryna V
authors.initials: KV
authors.lastname: Kotsarenko
authors.affiliation: Department of Medicinal Nanobiotechnology, Pirogov Russian State Medical University, Ostrovitianov str. 1, Moscow, 117997 Russia.
authors.forename: Vladimir P
authors.initials: VP
authors.lastname: Chekhonin
authors.affiliation: CNRS UMR8126, Institut de Cancérologie Gustave Roussy, Université Paris-Sud 11, Camille-Desmoulins str. 39, Villejuif, 94805 France.
authors.forename: Yegor S
authors.initials: YS
authors.lastname: Vassetzky
authors.affiliation: Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Zabolotnogo str. 150, Kiev, 03680 Ukraine.
authors.forename: Stanislav S
authors.initials: SS
authors.lastname: Avdieiev
authors.affiliation: Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Zabolotnogo str. 150, Kiev, 03680 Ukraine.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Dmitrenko
keywords: Aneuploidy; Chromosome instability; Drug resistance; Glioma; Heterogeneity; Karyotype