Human bone cell enzyme expression and cellular heterogeneity: Correlation of alkaline phosphatase enzyme activity with cell cycle

Abstract

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate‐resistant and ‐sensitive acid phosphatases, and non‐specific esterases were assayed kinetically and histo‐chemically. In asynchronous subconfluent cultures, < 15% of the cells stained positive and assayed activity was 0.04 IU/10⁶ cells/cm². After 1 week, the percent of alkaline phosphatase positive‐staining cells increased 5‐fold, while activity increased 10‐fold. Non‐specific esterases and tartrate‐sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate‐resistant acid phos‐phatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G₂ + 1/2 M of 4 h, and a G₁ of 25–30 h. In synchronous cultures induced by a thymidine‐aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G₁. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate‐sensitive acid phos‐phatase and non‐specific esterase levels were relatively stable throughout the cell cycle, while tartrate‐resistant acid phosphatase activity was not assavable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.