UNDEREXPRESSION OF THE 43 KDA INOSITOL POLY-PHOSPHATE 5-PHOSPHATASE IS ASSOCIATED WITH CELLULAR TRANSFORMATION

Abstract

In response to agonist stimulation the second messenger molecule inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) mobilises calcium from intracellular stores. The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses Ins(l,4,5)P3 in a signal terminating reaction. We have underexpressed the 43 kDa 5-phosphatase by stably transfecting NRK cells with the cDNA encoding the 43 kDa 5-phosphatase, cloned in the antisense orientation into the tetracycline-inducible expression vector pUHD10-3. Antisense-transfected cells demonstrated a greater than 50% reduction in Ins(l,4,5)P3 5-phosphatase activity in both the total cell homogenate and the detergent-soluble membrane fraction of the cell, as compared to cells transfected with vector alone. Unstimulated antisensetransfected cells showed a concomitant two fold increase in Ins(l,4,5)P and four fold increase in Ins(l,3,4,5)P levels. The basal intracellular calcium concentration of serum-deprived antisense-transfected cells (170 nM + 25) was increased 1.9 fold, compared to cells transfected with vector alone (90 nM + 25). In addition, antisense-transfected cell lines with basal intracellular cell calcium concentrations greater than 150 nM, were noted to have a significant decrease (5 fold) in total intracellular calcium stores. Cells underexpressing the 43 kDa 5-phosphatase demonstrated a transformed phenotype. Antisense-transfected cells grew at a 1.7 fold faster rate, reached confluence at higher density, and demonstrated increased [H]thymidine incorporation, compared to cells transfected with vector alone. Furthermore, antisense-transfected cells formed colonies in soft agar and tumours in nude mice. These studies support the contention that a decrease in cellular Ins(l,4,5)P3 5-phosphatase activity is associated with cellular transformation and implicates the 43 kDa inositol polyphosphate 5-phosphatase as a potential tumour suppressor gene.