title: Molecular characterization of acquired tolerance of tumor cells to picropodophyllin (PPP).
abstract: BACKGROUND
  Picropodophyllin (PPP) is a promising novel anti-neoplastic agent that efficiently kills tumor cells in vitro and causes tumor regression and increased survival in vivo. We have previously reported that PPP treatment induced moderate tolerance in two out of 10 cell lines only, and here report the acquired genomic and expression alterations associated with PPP selection over 1.5 years of treatment.
  
  
  RESULTS
  Copy number alterations monitored using metaphase and array-based comparative genomic hybridization analyses revealed largely overlapping alterations in parental and maximally tolerant cells. Gain/amplification of the MYC and PVT1 loci in 8q24.21 were verified on the chromosome level. Abnormalities observed in connection to PPP treatment included regular gains and losses, as well as homozygous losses in 10q24.1-q24.2 and 12p12.3-p13.2 in one of the lines and amplification at 5q11.2 in the other. Abnormalities observed in both tolerant derivatives include amplification/gain of 5q11.2, gain of 11q12.1-q14.3 and gain of 13q33.3-qter. Using Nexus software analysis we combined the array-CGH data with data from gene expression profilings and identified genes that were altered in both inputs. A subset of genes identified as downregulated (ALDH1A3, ANXA1, TLR4 and RAB5A) or upregulated (COX6A1, NFIX, ME1, MAPK and TAP2) were validated by siRNA in the tolerant or parental cells to alter sensitivity to PPP and confirmed to alter sensitivity to PPP in further cell lines.
  
  
  CONCLUSIONS
  Long-term PPP selection lead to altered gene expression in PPP tolerant cells with increase as well as decrease of genes involved in cell death such as PTEN and BCL2. In addition, acquired genomic copy number alterations were observed that were often reflected by altered mRNA expression levels for genes in the same regions.
authors.affiliation: Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, CMM L8:01, Stockholm, Sweden. Jamileh.Hashemi@ki.se
authors.forename: Jamileh
authors.initials: J
authors.lastname: Hashemi
authors.forename: Claire
authors.initials: C
authors.lastname: Worrall
authors.forename: Daiana
authors.initials: D
authors.lastname: Vasilcanu
authors.forename: Mårten
authors.initials: M
authors.lastname: Fryknäs
authors.forename: Luqman
authors.initials: L
authors.lastname: Sulaiman
authors.forename: Mohsen
authors.initials: M
authors.lastname: Karimi
authors.forename: Wen-Hui
authors.initials: WH
authors.lastname: Weng
authors.forename: Weng-Onn
authors.initials: WO
authors.lastname: Lui
authors.forename: Christina
authors.initials: C
authors.lastname: Rudduck
authors.forename: Magnus
authors.initials: M
authors.lastname: Axelson
authors.forename: Helena
authors.initials: H
authors.lastname: Jernberg-Wiklund
authors.forename: Leonard
authors.initials: L
authors.lastname: Girnita
authors.forename: Olle
authors.initials: O
authors.lastname: Larsson
authors.forename: Catharina
authors.initials: C
authors.lastname: Larsson
mesh_terms: D022981:Allelic Imbalance / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D002869:Chromosome Aberrations / Q000187:drug effects; D016000:Cluster Analysis; D055028:Comparative Genomic Hybridization; D056915:DNA Copy Number Variations / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005838:Genotype; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008677:Metaphase / Q000187:drug effects; D009369:Neoplasms / Q000235:genetics*; D011034:Podophyllotoxin / Q000031:analogs & derivatives* / Q000494:pharmacology; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results; D032681:Spectral Karyotyping
chemical_list: D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; C415032:picropodophyllin; D011034:Podophyllotoxin