K-Ras-independent effects of the farnesyl transferase inhibitor L-744,832 on cyclin B1/Cdc2 kinase activity, G2/M cell cycle progression and apoptosis in human pancreatic ductal adenocarcinoma cells

Abstract

Pancreatic ductal adenocarcinoma is a highly lethal malignancy that is resistant to traditional cytotoxic therapy. High rates of activating codon 12 K-Ras mutations in this disease have generated considerable interest in the therapeutic application of novel farnesyl transferase inhibitors (FTIs). However, a comprehensive analysis of the effects of FTI treatment on pancreatic cancer cells has not been performed. Treatment of five different human pancreatic cancer cell lines with FTI L-744,832 resulted in inhibition of anchorage-dependent growth, with wide variation in sensitivity among different lines. Effective growth inhibition by L-744,832 correlated with accumulation of cells with a tetraploid (4N) DNA content and high levels of cyclin B1/cdc2 kinase activity, implying cell cycle arrest downstream from the DNA damage-inducible G2/M cell cycle checkpoint. In addition, sensitive cell lines underwent apoptosis as evidenced by changes in nuclear morphology and internucleosomal DNA fragmentation. L-744,832 at a concentration of 1 microM additively enhanced the cytotoxic effect of ionizing radiation, apparently by overriding G2/M checkpoint activation. The effects of FTI treatment on cell growth and cell cycle regulation were associated with changes in posttranslational processing of H-Ras and N-Ras, but not K-Ras. The results confirm the potential therapeutic efficacy of FTI treatment in pancreatic cancer, and suggest that farnesylated proteins other than K-Ras may act as important regulators of G2/M cell cycle kinetics.

Figure 1

Inhibition of pancreatic cancer cell growth by FTI. Five different human pancreatic cancer cell lines were treated with escalating doses of the farnesyl transferase inhibitor L-744,832. Control cells were treated with 0.1% DMSO. Growth inhibition was determined by direct counting on days 1, 2, and 3 following initiation of treatment The y axis indicates cell number, expressed as per cent of same day DMSO-treated control cells. The x axis indicates time in days. Values represent mean±SEM of quadruplicate plates. Open triangles, L-744,832 100 nM. Open squares, L-744,832 500 nM. Closed inverted triangles, L-744,832 1 µM. Closed circles, L-744,832 10 µM. Closed triangles, L-744,832 25 µM. Closed squares, L-744,832 50 µM.

Figure 2

Effect of FTI on cell cycle kinetics in pancreatic cancer cell lines. Five different human pancreatic cancer cell lines were treated with either L-744,832 or 0.1% DMSO vehicle control. Cell cycle distribution was determined by propidium iodide staining and flow cytometry. (A) Cell cycle profiles of all five cell lines following treatment with 10 µM L-744,832 for 72 hours. Upper “a” panels indicate control cells. Lower “b” panels indicate FTI-treated cells. 1a and 1b, Aspc-1 cells. 2a and 2b, Bxpc-3 cells. 3a and 3b, Capan-2 cells. 4a and 4b, Cfpac-1 cells. 5a and 5b, Panc-1 cells. (B) Accumulation of cells with 4N DNA population following FTI treatment. For each cell line, the fraction of cells with 4N DNA population (G2/M) was determined following treatment with 0.1% DMSO (white bars) or with 10 µM L-744,832 (black bars) for 72 hours. Values represent mean±SEM of three independent experiments for each condition, with each experiment involving cell cycle analysis of 10,000 cells. (C) Dose-dependent accumulation of cells with 4N DNA population following FTI treatment. Values indicate fraction of cells with 4N DNA population (G2/M) in single representative experiment. Black bars, 0.1% DMSO. White bars, L-744,832 100 nM. Horizontal hatched bars, L-744,832 500 nM. Upsloping hatched bars, L-744,832 1 µM. Downsloping hatched bars, L-744,832 10 µM. Light grey bars, L-744,832 25 µM. Dark grey bars, L-744,832 50 µM.

Figure 3

Induction of apoptosis by FTI in pancreatic cancer cell lines. Cells were treated with either L-744,832 (10 µM) or O.1% DMSO vehicle control for 72 hours. Nuclear morphology was assessed by propidium iodide staining, and the induction of apoptosis was confirmed by TUNEL assay. (A–D) Panc-1 cells. (E–H) Cfpac-1 cells. (A), (B), (E), (F) Propidium iodide staining. (C), (D), (G), (H) TUNEL labeling. (A), (C), (E), (G) Cells treated with 0.1% DMSO vehicle control. (B), (D), (F), (G) Cells treated with 10 µM L-744,832. Note nuclear fragmentation and high frequency of TUNEL positivity in FTI-treated Panc-1 cells. Apoptotic indices from replicate experiments are provided in Table 1.

Figure 4

Effect of FTI on growth inhibitory effects of ionizing radiation in Panc-1 cells. Cells were exposed to escalating doses of ionizing radiation (0 to 8 Gy) and immediately treated with 0.1% DMSO or escalating concentrations of L-744,832 (0 to 10 µM). Values indicate mean±SEM of three independent experiments. (A) Cell number on day 2 following radiation with or without simultaneous L-744,832 treatment. Y axis indicates cell number as percentage of untreated control. White bars, 0 Gy. Light grey bars, 1 Gy. Dark grey bars, 2 Gy. Black bars, 4 Gy. Horizontal hatched bars, 6 Gy. Diagonal hatched bars, 8 Gy. (B) Cell number as a function of time following radiation with or without simultaneous treatment with 1 µM L-744,832. Y axis indicates cell number as percentage of day 2 untreated control. X axis indicates time in days. Closed squares, 0 Gy+0.1% DMSO. Closed triangles, 0 Gy+1 µM L-744,832. Open squares, 4 Gy+0.1% DMSO. Open triangles, 4 Gy+1 µM L-744,832.

Figure 5

Effect of FTI on cyclin B1/cdc2 kinase activity in irradiated and nonirradiated pancreatic cancer cells. Autoradiographs depict incorporation of radioactive phosphate into histone HI substrate. Bar graphs depict quantified kinase activity as mean±SEM of three independent experiments. (A) Effect of FTI on cyclin B1/cdc2 kinase activity in nonirradiated pancreatic cancer cell lines. Cells were treated with either L-744,832 (10 µM) or 0.1% DMSO vehicle control for 72 hours. For each cell line, data are expressed as percentage of DMSO-treated control. White bars, DMSO-treated control conditions. Black bars, L-744,832-treated cells. (B) Effect of escalating concentrations of FTI on cyclin B1/cdc2 kinase activity following ionizing radiation (IR). Panc-1 cells were exposed to 0 Gy or 8 Gy of ionizing radiation, immediately treated with 0.1% DMSO or escalating concentrations of L-744,832, and harvested 8 hours later for measurement of kinase activity. Data are expressed as percentage of nonirradiated DMSO-treated control cells. White bars, nonirradiated cells. Black bars, irradiated cells. (C) Temporal changes in cyclin B1/cdc2 kinase activity following ionizing radiation in cells treated with and without FTI. Panc-1 cells were exposed to 0 Gy versus 8 Gy of ionizing radiation, and immediately treated with 0.1% DMSO or 1 µM L-744,832. Kinase activity was determined at indicated time points. White bars, DMSO-treated cells. Black bars, L-744,832-treated cells.

Figure 6

Effect of FTI on subcellular localization and posttranslational processing of ras proteins. (A) Pan-ras immunoreactivity in Panc-1 cells treated with 0.1% DMSO for 72 hours. Note predominant staining of cytoplasmic membrane. (B) Pan-ras immunoreactivity in Panc-1 cells treated with 10 µM L-744,832 for 72 hours. Increased cytoplasmic staining is noted. (C) Immunoblot analysis of FTI-induced changes in posttranslational processing of Ras proteins. Cells were treated with 0.1% DMSO versus 10 µM L-744,832 for 72 hours. “U” indicates unprocessed upper band. “P” indicates processed lower band. Results are representative of two independent experiments.