VARIATION IN 1,25-DIHYDROXYVITAMIN-D3 REGULATION OF PROLIFERATION AND ALKALINE-PHOSPHATASE ACTIVITY IN LATE-PASSAGE RAT OSTEOBLASTIC CELL-LINES

The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], 1,25-dihydroxy-16ene-23yne-vitamin D3[1,25(OH)2-16ene-23yne-D3] (a synthetic analog) and retinoic acid on proliferation, protein synthesis, and alkaline phosphatase activity and mRNA were compared in two late-passage (P > 70) clonal rat osteoblastic cell lines (G2 and C12) in order to characterize variations in the basal and hormonally-regulated phenotypes. All agents inhibited proliferation (measured as cell number after 3 days of treatment) in late-passage (P > 70) G2 and C12 cells without inhibiting the rate of protein synthesis ([H-3]leucine incorporation into TCA-precipitable protein) during the last 18 h of incubation. Basal and hormone-treated alkaline phosphatase activities were lower in late-passage G2 and C12 cells than those previously reported for early-passage G2 and C12 cells. 1,25(OH)2D3 and 1,25(OH)2-16ene-23yne-D3 up-regulated alkaline phosphatase activity in late-passage C12 cells and down-regulated it in late-passage G2 cells. The direction of these regulatory changes in late-passage cells was opposite to that reported for early passages of these clones, and changes were related to the levels of tissue-unspecific alkaline phosphatase mRNA normalized for actin mRNA. Effects of 1,25(OH)2D3 or 1,25(OH)2-16ene-23yne-D3 and retinoic acid were not additive, suggesting a competitive mechanism of action. It appears that increased sensitivity to the antiproliferative effects of regulatory hormones and defects in proliferation and specialization of the osteoblast are observed with increasing passage number in vitro in two model osteoblastic cell lines (G2 and C12).