Suppression of cyclic GMP-specific phosphodiesterase 5 promotes apoptosis and inhibits growth in HT29 cells

Phosphodiesterase 5 (PDE5) is a major isoform of cGMP phosphodiesterase in a variety of human tumor cell lines and plays a key role in regulating intracellular cGMP concentrations ([cGMP]i). Here, we demonstrate that suppression of PDE5 gene expression by antisense pZeoSV2/ASP5 plasmid transfection results in a sustained increase in [cGMP]i, growth inhibition, and apoptosis in human colon tumor HT29 cells. With stable transfection, antisense transcripts exhibited a specific suppression in PDE5 activity, mRNA levels, and a 93 kDa hPDE5A1 protein. In cloned antisense cells, prolongation of the cell growth doubling times correlate positively with suppressed PDE5 activity and increased [cGMP]i. The growth inhibition in PDE5 antisense clones is due to an increased apoptotic rate and delayed cell-cycle progression. These results corroborate previous findings with the PDE5 inhibitor exisulind and its derivatives showing that sustained [cGMP]i induces apoptosis and growth inhibition in tumor cells. Furthermore, an inducible mitotic inhibitor p21(WAF1)/(CIP1) has been found to account for the delay of cell-cycle progression in PDE5 antisense clones at G(2)/M phase. A proteolytic cleavage of p21(WAF1)/(CIP1) in the antisense clones is also increased at the later stage of serum stimulation. The protein kinase G (PKG) inhibitor, KT5823, can prevent the cleavage of p21 (WAF1 /CIP). These data substantiate a pivotal role for PDE5 as a modulator of apoptosis and cell-cycle progression for human carcinoma via a mechanism involving the activation of [cGMP](i)/PKG signaling pathways. (C) 2004 Wiley-Liss, Inc.