Expression and nature of the alkaline phosphatase gene in cultured osteosarcoma cells.
The molecular mechanism for the differences in specific activity of alkaline phosphatase in six human osteosarcoma cell lines was investigated. Five of the lines expressed only the tissue-non-specific or liver/bone/kidney isoenzyme of alkaline phosphatase. The sixth line had the lowest levels of alkaline phosphatase and this was determined to be a mixture of liver/bone/kidney isoenzyme and at least one other form. The mRNA of liver/bone/kidney alkaline phosphatase was identified by Northern analysis in the three cell lines that expressed the largest amount of alkaline phosphatase catalytic activity. This mRNA was indistinguishable in size from that seen in control mRNA from normal kidney (2.5 kb). Southern analysis demonstrated that EcoRI or HindIII restriction fragment patterns and the intensity of the bands, of the liver/bone/kidney alkaline phosphatase gene in the osteosarcoma cell lines were identical to that of the control DNA from normal peripheral blood leukocytes. Thus, the gene coding for liver/bone/kidney alkaline phosphatase appears to be intact in all of these osteosarcoma cells and it is unlikely that rearrangement, deletion or amplification of the gene is responsible for its activation or inactivation. Slot blot analysis revealed varying amounts of the transcripts of the liver/bone/kidney isoenzyme in each of the cell lines. The best fit line of a plot of the log of the level of mRNA of alkaline phosphatase vs. the log of the specific activity of liver/bone/kidney alkaline phosphatase was constructed. This gave a Pearson correlation coefficient of 0.92 (P < 0.008), demonstrating a significant relationship between the two variables. It is likely that the regulation of alkaline phosphatase activity is at the transcriptional process rather than the translational or post-translational processes and that the specific activity of the enzyme may be controlled by the amount of steady-state mRNA of the liver/bone/kidney isoenzyme.
Stinson
R A
RA
Division of Medical Laboratory Science, University of Alberta, Edmonton, Canada.
Thacker
J D
JD
Lin
C C
CC
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D015152:Blotting, Northern; D015139:Blotting, Southern; D001859:Bone Neoplasms / Q000201:enzymology* / Q000235:genetics*; D002384:Catalysis; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D012516:Osteosarcoma / Q000201:enzymology* / Q000235:genetics*; D012313:RNA / Q000032:analysis; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D012313:RNA; D000469:Alkaline Phosphatase