aphidicolin and Fibrosarcoma

A combination of four genetic suppressor elements (GSEs), two of which are derived from putative transcriptional regulators, was previously found to increase resistance to drugs inhibiting DNA replication in HT1080 fibrosarcoma cells. In the present study, two GSE-transduced cell lines, isolated with and without cytotoxic selection, were found to be resistant to a diverse group of DNA-interactive agents, including aphidicolin, hydroxyurea, cytarabine, etoposide, doxorubicin, and mafosfamide. Changes in gene expression associated with GSE-induced drug resistance were analyzed by cDNA array hybridization and reverse transcription-PCR. Twenty genes were found to be up-regulated in both of the resistant cell lines. These include genes involved in DNA replication and repair (e.g., PCNA, XRCC1, B-MYB, and GADD45), transcriptional regulators associated with stress response, and cell cycle checkpoint control (e.g., YB-1, DBPA, and ATF4), and genes for signal transduction proteins (e.g., protein tyrosine phosphatase 1B and regulatory subunits alpha and beta of cAMP-dependent protein kinase). The observed changes in gene expression may play a role in pleiotropic resistance to different classes of DNA-targeting drugs.

Topics: Antineoplastic Agents; Aphidicolin; Cell Cycle; DNA Repair; DNA Replication; DNA, Neoplasm; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fibrosarcoma; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Suppressor; Heat-Shock Response; Humans; Proliferating Cell Nuclear Antigen; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription Factors; Transduction, Genetic; Tumor Cells, Cultured

Post-synthetic enzymatic hypermethylation of DNA was induced in hamster fibrosarcoma cells by the DNA synthesis inhibitors cytosine arabinoside, hydroxyurea and aphidicolin. This effect required direct inhibition of DNA polymerase alpha or reduction in deoxynucleotide pools and was not specific to a single cell type. At equivalently reduced levels of DNA synthesis, neither cycloheximide, actinomycin D nor serum deprivation affected DNA methylation in this way. The topoisomerase inhibitors nalidixic acid and novobiocin caused significant hypomethylation indicating that increased 5-mCyt content was not a necessary consequence of DNA synthesis inhibition. The induced hypermethylation occurred predominantly in that fraction of the DNA synthesized in the presence of inhibitor; was stable in the absence of drug; was most prominent in low molecular weight DNA representing sites of initiated but incomplete DNA synthesis; and occurred primarily within CpG dinucleotides, although other dinucleotides were overmethylated as well. Drug-induced CpG hypermethylation may be capable of silencing genes, an effect which may be relevant to the aberrantly expressed genes characteristic of neoplastic cells.

Topics: Animals; Aphidicolin; Base Sequence; Cell Line; Chromatography, High Pressure Liquid; Cricetinae; Cycloheximide; Cytarabine; Dactinomycin; Diterpenes; DNA; DNA Polymerase II; DNA Replication; Fibrosarcoma; Hydroxyurea; Methylation; Nalidixic Acid; Novobiocin