Effect of Cyclin D1 Overexpression on Drug Sensitivity in a Human Fibrosarcoma Cell Line

• Published in: JNCI : Journal of the National Cancer Institute 1996-09, Vol.88 (18), p.1269-1275
• Main Authors: Hochhauser, Daniel, Schnieders, Barbara, Ercikan-Abali, Emine, Gorlick, Richard, Muise-Helmericks, Robin, Li, Wei-Wei, Fan, Jianguo, Banerjee, Debabrata, Bertino, Joseph R.
• Format: Article
• Language: English
• Subjects: Antimetabolites, Antineoplastic - pharmacology; Antineoplastic agents; Antineoplastic Agents - pharmacology; Biological and medical sciences; Blotting, Northern; Blotting, Western; Cancer; Cells; Chemotherapy; Cyclin D1; Cyclins - biosynthesis; Cyclins - drug effects; Cyclins - genetics; Drug Resistance, Neoplasm; Fibrosarcoma - drug therapy; Fibrosarcoma - metabolism; Flow Cytometry; Gene Expression Regulation, Neoplastic; General aspects; Humans; Medical research; Medical sciences; Methotrexate - pharmacology; Neomycin - pharmacology; Oncogene Proteins - biosynthesis; Oncogene Proteins - drug effects; Oncogene Proteins - genetics; Pharmacology. Drug treatments; Phosphorylation; Protein Synthesis Inhibitors - pharmacology; Proteins; Retinoblastoma Protein - drug effects; Retinoblastoma Protein - metabolism; RNA, Messenger - drug effects; Tetrahydrofolate Dehydrogenase - drug effects; Tetrahydrofolate Dehydrogenase - genetics; Transfection; Tumor Cells, Cultured; Up-Regulation
• ISSN: 0027-8874; 1460-2105
• DOI: 10.1093/jnci/88.18.1269

Background Alterations in the expression of genes that control the cell cycle may be of critical importance in determining the sensitivity of cells and tumors to drugs (chemosensitivity) and radiation. Mutations and deletions of the p53 tumor suppressor gene in cell lines and tumors are associated with resistance to a variety of DNA-damaging agents. The effects of alterations in the cyclin genes and their products on drug action have not been studied. One of these genes, cyclin D1, is expressed in early G1 phase, and its protein product, together with the cyclin-dependent kinases CDK4 and CDK6, mediates the phosphorylation and functional inactivation of the retinoblastoma protein (pRb). Elevated levels of expression of cyclin D1 protein have been found in a variety of cancers, including breast cancer, head and neck cancer, non-small-cell lung cancer, and mantle cell lymphomas. Purpose This study was conducted to investigate the effect of increased expression of cyclin D1 protein on the chemosensitivity profile of a human fibrosarcoma cell line. Methods Expression plasmids containing either the neomycin-resistance gene and the complementary DNA sequence encoding human cyclin D1 or the neomycin-resistance gene only (control) were transfected by lipofection into the human HT1080 fibrosarcoma cell line, and cell colonies resistant to the antibiotic neomycin (G418) were isolated. Cyclin D1 messenger RNA (mRNA) and protein levels were measured by ribonuclease protection and western blot analyses, respectively. Dihydrofolate reductase (DHFR) mRNA and protein levels were measured by northern blot and western blot analyses, respectively. The phosphorylation status of pRb was assessed by western blot analysis. Cell cycle analysis was performed by use of the technique of fluorescence-activated cell sorting. Cytotoxicity assays were carried out by use of the sulforhodamine blue assay. Results Of the 16 cyclin D1-transfected cell clones that were isolated, four were randomly selected for further study. Two cell clones expressed high levels of cyclin D1 mRNA and protein as compared with control cells transfected with plasmids containing the neomycin-resistance gene only. A relative increase in the phosphorylated form of pRb in cells expressing high versus low levels of cyclin D1 was also revealed by western blot analysis. There was an increased fraction of cells in the S and G2 phases of the cell cycle among cells expressing higher levels of cyclin D1. Transfectants wit