5-Lipoxygenase has been expressed in a mammalian
osteosarcoma cell line transfected with the cloned
cDNA for human leukocyte
5-lipoxygenase. Two clonal cell lines derived from the transfected cells expressed the enzymatic activity. When incubated with
arachidonic acid (100 microM), the major
5-lipoxygenase products of 10,000 X g supernatants from these cells were
5-hydroxyeicosatetraenoic acid (5-HETE), and the nonenzymatic hydrolysis products of
leukotriene (LT)A4. The ratio of
5-HETE to LT (between 6:1 and 9:1) was similar to that observed in leukocyte supernatants. Furthermore, incubation of 10,000 X g supernatants from the transfected cells with 5-hydroperoxyeicosatetraenoic
acid (5-HPETE) (75 microM) resulted in the synthesis of
LTA4 hydrolysis products. Control
osteosarcoma cell supernatants produced no
5-HETE or LT from
arachidonic acid or
5-HPETE. Maximal activity of the expressed
enzyme required Ca2+,
ATP, and two cellular stimulatory factors prepared from human leukocytes. Immunoblot analysis of supernatants from the
osteosarcoma cell clones revealed an immunoreactive 80,000-dalton band that was indistinguishable from the band observed in leukocyte supernatants. Therefore, the expressed
enzyme was functional and exhibited characteristics that were identical to those of human leukocyte
5-lipoxygenase. When intact transfected
osteosarcoma cells were challenged with
ionophore A 23187, no
5-lipoxygenase products were formed. If
arachidonic acid was added along with the
ionophore, the cells synthesized
5-HETE and the nonenzymatic hydrolysis products of
LTA4. These results verify that the
cDNA used to transfect the
osteosarcoma cells encodes for
5-lipoxygenase. Furthermore, these studies offer independent evidence that this single
protein possesses both
5-lipoxygenase and
LTA4 synthase activity, as has been reported previously from
enzyme purification data.