Inhibition of the cyclin D1/E2F pathway by PCA-4230, a potent repressor of cellular proliferation

Abstract

1. Tight control of cellular growth is essential to ensure normal tissue patterning and prevent pathological responses. Excessive vascular smooth muscle cell (VSMC) proliferation is associated with the pathophysiology of atherosclerosis and restenosis post-angioplasty. Thus, drug targeting of pathological VSMC growth may be a suitable therapeutic intervention in vascular proliferative diseases. 2. In the present study, we investigated the mechanisms underlying VSMC growth arrest induced by the pharmacological agent PCA-4230. Addition of PCA-4230 to cultured VSMCs blocked the induction of cyclin D1 and cyclin A expression normally seen in serum-restimulated cells. Moreover, PCA-4230 inhibited cyclin-dependent kinase 2 (CDK2) activity and abrogated hyperphosphorylation of the retinoblastoma (Rb) gene product. Similarly, PCA-4230-dependent growth arrest of transformed cell lines correlated with reduced level of cyclin D1 protein and inhibition of CDK2 activity. Consistent with these findings, PCA-4230 repressed serum-inducible cyclin A promoter activity, and overexpression of either cyclin D1 or E2F1 efficiently circumvented this inhibitory effect. Importantly, adenovirus-mediated overexpression of E2F1 restored S-phase entry in PCA-4230-treated VSMCs, demonstrating that PCA-4230 represses cyclin A gene expression and VSMC growth via inhibition of the cyclin D1/E2F pathway. 3. Because of its ability to inhibit the growth of human VSMCs and transformed cell lines, future studies are warranted to assess whether PCA-4230 may be a suitable therapeutic intervention for the treatment of hyperproliferative disorders, including cardiovascular disease and cancer.

Figure 1

PCA-4230 inhibits serum-inducible proliferation of rat and human VSMCs. Results represent the mean±s.e.mean of three independent measurements. (A) The rat E19P cell line and primary human VSMCs were plated into 6-well dishes and maintained with medium containing 10% FBS plus vehicle or PCA-4230. Cells were trypsinized at different times and cell number was determined with a haemocytometer. (B, C) Cells were starved for 3 days in medium containing 0.5% FBS. After pretreatment with vehicle or PCA-4230, cells were stimulated with 10% FBS plus vehicle or PCA-4230. Cells were harvested for FACS analysis after 16 h of serum stimulation.

Figure 2

PCA-4230 inhibits proliferation of transformed cells. Cells were maintained in medium containing 10% FBS plus vehicle or PCA-4230. Results represent the mean±s.e.mean of three independent measurements. (A) U2OS and MCF7 cells were plated in 6-well dishes. After 3 days, cells were trypsinized and cell number was determined with a haemocytometer. (B) MEF-Myc cells growing on 6-well dishes were trypsinized at different times and cell number was determined with a haemocytometer. (C) Effect of PCA-4230 on the incorporation of ³H-thymidine into MEF-Myc cells (see Methods for details).

Figure 3

PCA-4230 inhibits CDK2 activity. Subconfluent E19P and MEF-Myc cells maintained in 10% FBS were treated for 15 h with vehicle or the indicated amounts of PCA-4230. (A) Cell lysates were assayed for CDK2 activity using histone H1 as substrate. (B) PI3K activity in E19P lysates using L-α-phosphatidylinositol as substrate. PIP: L-α-phosphatidylinositol phosphate.

Figure 4

Western blot analysis of cell cycle regulatory proteins in PCA-4230-treated cells. Control cultures were exposed to vehicle. (A, C) E19P cells were starvation-synchronized for 3 days in medium containing 0.5% FBS. One-and-a-half hours before serum-restimulation, 50 μM PCA-4230 was added and treatment continued throughout the period of serum restimulation. The antibody used in each blot is shown. pRb: hypophosphorylated Rb; ppRb: hyperphosphorylated Rb. (B) Cyclin D1 expression in asynchronously growing MEF-Myc cells. (D) p27 expression in asynchronously growing MCF7, U2OS and E19P cells.

Figure 5

PCA-4230 inhibits serum-inducible cyclin A promoter activity in VSMCs. E19P cells were cotransfected with 2 μg of a luciferase reporter gene driven by the human cyclin A promoter and 0.5 μg of a control plasmid encoding for alkaline phosphatase. Results are expressed as the ratio luciferase/alkaline phosphatase. Bars represent the mean±s.e.mean of three independent transfections. Control cells were treated with vehicle. Results are referred to the activity seen in control untreated cells (=100%). (A) Transfected cells were maintained in 0.5% FBS for 2 days. Cells were then harvested to determine basal cyclin A promoter activity in serum-starved cells (first bar), or were pretreated for 2 h with vehicle or PCA-4230 and then serum-restimulated overnight. (B) Cells were maintained in 10% FBS throughout the experiment.

Figure 6

Ectopic overexpression of either cyclin D1 or E2F1 can overcome the inhibitory effect of PCA-4230 on cyclin A promoter activity. Cells were treated as in Figure 5, except that cultures were maintained throughout the experiment in 10% FBS with vehicle or with 50 μM PCA-4230. Results are expressed as the ratio luciferase/alkaline phosphatase. Bars represent the mean±s.e.mean of three independent transfections. Results are referred to the activity seen in control untreated cells (=100%). Cells were cotransfected with CMV-Cyclin D1 (0.1 μg per transfection, A), or with CMV-E2F1 (0.2 μg per transfection, B).

Figure 7

Adenovirus-mediated overexpression of E2F1 restores S-phase entry in PCA-4230-treated VSMCs. E19P cells were maintained for 3 days in 0.5% FBS. Cells were then harvested for FACS analysis to determine basal activity (first bar), or pretreated with vehicle or 50 μM PCA-4230 followed by 16 h of stimulation with 10% FBS. When indicated, starved cells were infected with replication-defective Ad-E2F1 or Ad-βgal at different multiplicities of infection (MOI) during the last 8 h of serum starvation. Results represent the mean±s.e.mean of three experiments.