Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with cellular transformation.
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.
Speed
C J
CJ
Department of Medicine, Monash Medical School, Box Hill Hospital, Melbourne, Australia.
Little
P J
PJ
Hayman
J A
JA
Mitchell
C A
CA
D016475:3T3 Cells; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D016147:Genes, Tumor Suppressor; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D007668:Kidney / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D051381:Rats; D014162:Transfection
D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases; D002118:Calcium