The vitamin K-dependent carboxylation system in human osteosarcoma U2-OS cells. Antidotal effect of vitamin K1 and a novel mechanism for the action of warfarin

Abstract

An osteoblast-like human osteosarcoma cell line (U2-OS) has been shown to possess a vitamin K-dependent carboxylation system which is similar to the system in human HepG2 cells and in liver and lung from the rat. In an 'in vitro' system prepared from these cells, vitamin K1 was shown to overcome warfarin inhibition of gamma-carboxylation carried out by the vitamin K-dependent carboxylase. The data suggest that osteoblasts, the cells involved in synthesis of vitamin K-dependent proteins in bone, can use vitamin K1 as an antidote to warfarin poisoning if enough vitamin K1 can accumulate in the tissue. Five precursors of vitamin K-dependent proteins were identified in osteosarcoma and HepG2 cells respectively. In microsomes (microsomal fractions) from the osteosarcoma cells these precursors revealed apparent molecular masses of 85, 78, 56, 35 and 31 kDa. When osteosarcoma cells were cultured in the presence of warfarin, vitamin K-dependent 14C-labelling of the 78 kDa precursor was enhanced. Selective 14C-labelling of one precursor was also demonstrated in microsomes from HepG2 cells and from rat lung after warfarin treatment. In HepG2 cells this precursor was identified as the precursor of (clotting) Factor X. This unique 14C-labelling pattern of precursors of vitamin K-dependent proteins in microsomes from different cells and tissues reflects a new mechanism underlying the action of warfarin.

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“…Transient transfection studies were performed in U2‐OS and 293 cells using the TransFast Reagent (Promega, Madison, WI). U2‐OS cell is an osteosarcoma cell line that could be used as an in vitro model system for bone formation during development [Wallin et al, 1990]. The 293 cell line is derived from a human embryonic kidney that is widely used as a tool for exogenous gene expression [Graham et al, 1977].…”