Abstract
Recently we have shown that in fibroblasts (NIH 3T3 and Rat-1 cells) inhibition of protein geranylgeranylation leads to a G0/G1 arrest, whereas inhibition of protein farnesylation does not affect cell cycle distribution. Here we demonstrate that in human tumor cells the geranylgeranyltransferase-I (GGTase-I) inhibitor GGTI-298 blocked cells in G0/G1, whereas the farnesyltransferase (FTase) inhibitor FTI-277 showed a differential effect depending on the cell line. FTI-277 accumulated Calu-1 and A-549 lung carcinoma and Colo 357 pancreatic carcinoma cells in G2/M, T-24 bladder carcinoma, and HT-1080 fibrosarcoma cells in G0/G1, but had no effect on cell cycle distribution of pancreatic (Panc-1), breast (SKBr 3 and MDAMB-231), and head and neck (A-253) carcinoma cells. Furthermore, treatment of Calu-1, Panc-1, Colo 357, T-24, A-253, SKBr 3, and MDAMB-231 cells with GGTI-298, but not FTI-277, induced the protein expression levels of the cyclin-dependent kinase inhibitor p21WAF. HT-1080 and A-549 cells had a high basal level of p21WAF, and GGTI-298 did not further increase these levels. Furthermore, GGTI-298 also induces the accumulation of large amounts of p21WAF mRNA in Calu-1 cells, a cell line that lacks the tumor suppressor gene p53. There was little effect of GGTI-298 on the cellular levels of another cyclin- dependent kinase inhibitor p27KIP as well as cyclin E and cyclin D1. These results demonstrate that GGTase-I inhibitors arrest cells in G0/G1 and induce accumulation of p21WAF in a p53-independent manner and that FTase inhibitors can interfere with cell cycle events by a mechanism that involves neither p21WAF nor p27KIP. The results also point to the potential of GGTase-I inhibitors as agents capable of restoring growth arrest in cells lacking functional p53.
Highlights
We have shown that in fibroblasts (NIH 3T3 and Rat-1 cells) inhibition of protein geranylgeranylation leads to a G0/G1 arrest, whereas inhibition of protein farnesylation does not affect cell cycle distribution
GGTI-298 Arrests Human Tumor Cells in the G0/G1 Phase of the Cell Cycle—We have recently shown that the GGTase-I inhibitor GGTI-298 arrested NIH 3T3 and Rat-1 cells in the G0/G1 phase of the cell cycle, whereas the FTase inhibitor FTI-277 had no effect on the cell cycle distribution of these cells
GGTI-298 and lovastatin led to a G0/G1 arrest in Calu-1, T-24, and A-253 cells
Summary
Synthesis of CAAX Peptidomimetics—The FTase-specific peptidomimetic FTI-277 and the GGTase-I-specific peptidomimetic GGTI-298 were synthesized as described previously [9, 10, 34]. 100-mm Petri dishes, treated with vehicle or inhibitors every 24 h for 48 h, harvested, and lysed in HEPES lysis buffer as described [13]. Northern Blotting—Calu-1 cells were grown to 30% confluency and treated with vehicle or inhibitors for 48 h. RNA aliquots (10 –15 g) were electrophoresed on a 1% agarose gel, transferred to nylon hybridization membranes (GeneScreen, NEN Life Science Products) and blotted with radiolabeled p21WAF and cyclophilin cDNA probes [32]. Flow Cytometry—Cells were plated in 100-mm Petri dishes and treated with inhibitors or vehicle as described for the Western blot procedure, and nuclei were prepared as described [38]. Cells were harvested by gentle scraping, pelleted by low speed centrifugation, resuspended in 200 l of citrate buffer (250 mM sucrose, 40 mM trisodium citrate, 5% Me2SO, pH 7.6), and frozen at Ϫ80 °C. The proportions of cell in G0/G1, S, and G2/M phase were calculated from their DNA histogram using the BDIS Consort software package (Ver. 3.0, Becton-Dickinson)
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