| Abstrakt: |
The 43 kDa inositol polyphosphate 5‐phosphatase (5‐phosphatase) hydrolyses the second messenger molecules inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4]. We have underexpressed the 43 kDa 5‐phosphatase by stably transfecting normal rat kidney cells with the cDNA encoding the enzyme, cloned in the antisense orientation into the tetracycline‐inducible expression vector pUHD10–3. Antisense‐transfected cells demonstrated a 45% reduction in Ins(1,4,5)P3 5‐phosphatase activity in the total cell homogenate upon withdrawal of tetracycline, and an approximately 80% reduction in the detergent‐soluble membrane fraction of the cell, as compared with antisense‐transfected cells in the presence of tetracycline. Unstimulated antisense‐transfected cells showed a concomitant 2‐fold increase in Ins(1,4,5)P3 and 4‐fold increase in Ins(1,3,4,5)P4 levels. The basal intracellular calcium concentration of antisense‐transfected cells (170 +/− 25 nM) was increased 1.9‐fold, compared with cells transfected with vector alone (90 +/− 25 nM). 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 [3H]thymidine incorporation compared with 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 Ins(1,4,5)P3 5‐phosphatase activity is associated with cellular transformation. |
