EFFECT OF CYCLIN D1 OVEREXPRESSION ON DRUG-SENSITIVITY IN A HUMAN FIBROSARCOMA CELL-LINE

Background: Alterations in the expression of genes that control the ce ll cycle may be of critical importance in determining the sensitivity of cells and tumors to drugs (chemosensitivity) and radiation. Mutatio ns and deletions of the p53 tumor suppressor gene in cell lines and tu mors are associated with resistance to a variety of DNA-damaging agent s. The effects of alterations in the cyclin genes and their products o n drug action have not been studied. One of these genes, cyclin D1, is expressed in early G(1) phase, and its protein product, together with the cyclin-dependent kinases CDK4 and CDK6, mediates the phosphorylat ion and functional inactivation of the retinoblastoma protein (pRb). E levated levels of expression of cyclin D1 protein have been found in a variety of cancers, including breast cancer, head and neck cancer, no n-small-cell lung cancer, and mantle cell lymphomas. Purpose: This stu dy was conducted to investigate the effect of increased expression of cyclin D1 protein on the chemosensitivity profile of a human fibrosarc oma cell line. Methods: Expression plasmids containing either the neom ycin-resistance gene and the complementary DNA sequence encoding human cyclin D1 or the neomycin-resistance gene only (control) were transfe cted by lipofection into the human HT1080 fibrosarcoma cell line, and cell colonies resistant to the antibiotic neomycin (G418) were isolate d. Cyclin D1 messenger RNA (mRNA) and protein levels were measured by ribonuclease protection and western blot analyses, respectively. Dihyd rofolate reductase (DHFR) mRNA and protein levels were measured by nor thern blot and western blot analyses, respectively. The phosphorylatio n status of pRb was assessed by western blot analysis. Cell cycle anal ysis was performed by use of the technique of fluorescence-activated c ell sorting. Cytotoxicity assays were carried out by use of the sulfor hodamine blue assay. Results: Of the 16 cyclin D1-transfected cell clo nes that were isolated, four were randomly selected for further study. Two cell clones expressed high levels of cyclin D1 mRNA and protein a s compared with control cells transfected with plasmids containing the neomycin-resistance gene only. A relative increase in the phosphoryla ted form of pRb in cells expressing high versus low levels of cyclin D 1 was also revealed by western blot analysis. There was an increased f raction of cells in the S and G(2) phases of the cell cycle among cell s expressing higher levels of cyclin D1. Transfectants with increased cyclin D1 expression also had increased DHFR mRNA and protein expressi on. Cytotoxicity assays revealed a statistically significant (P<.01) i ncrease 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 cyta rabine. Conclusion: Alterations in the expression of cyclin D1 led to altered cell cycle distribution in a human sarcoma cell line. The asso ciated 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 g enes may differ in their effects on cytotoxicity. It will be important to determine the effects of alterations of other cell cycle regulator y genes on the responses of cells to specific classes of drugs. Tumors with overexpression of cyclin D1 may be relatively refractory to meth otrexate treatment.