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 with increased cyclin D1 expression also had increased DHFR mRNA and protein expression. Cytotoxicity assays revealed a statistically significant (P < .01) increase in resistance to methotrexate in cells expressing high levels of cyclin D1 compared with cells expressing lower levels. There was no difference in resistance to doxorubicin, paclitaxel (Taxol), and cytarabine.
Conclusion: Alterations in the expression of cyclin D1 led to altered cell cycle distribution in a human sarcoma cell line. The associated increase in DHFR expression resulted in increased resistance to methotrexate but had no effect on other classes of anticancer agents.
Implications: These results indicate that alterations in cell cycle genes may differ in their effects on cytotoxicity. It will be important to determine the effects of alterations of other cell cycle regulatory genes on the responses of cells to specific classes of drugs. Tumors with overexpression of cyclin D1 may be relatively refractory to methotrexate treatment.