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Evidence for Thromboxane Biosynthesis in Established Cell Lines Derived from Human Lung Adenocarcinomas¹

Program Development Research Group, Development Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701-1013

Thromboxane B₂ (TxB₂) is the stable nonenzymatic hydrolysis product of thromboxane A₂, a substance implicated in the initiation of the facilitative role of thrombocytes in the metastatic process. TxB₂ was isolated from protein-free culture medium of cell lines Calu-3, Calu-6, A549, and A549/Asc-1, derived from human lung adenocarcinomas. TxB₂ and other 20-carbon fatty acid cyclooxygenase products synthesized from exogenous and endogenous arachidonic acid were identified by their characteristic retention indices and fragmentation of electron-capture derivatives of unlabeled and deuterium-labeled products during combined capillary gas chromatography-mass spectrometry. TxB₂ comprised 2 to 6% of 20-carbon fatty acid cyclooxygenase products biosynthesized from endogenous arachidonic acid in calcium ionophore A23187-stimulated Calu-6 and A549/Asc-1 cells and 16 to 25% of these products in Calu-3 and A549 cells. The addition of 10^-5 M exogenous arachidonic acid to the cultured cells resulted in a 2- to 3-fold increase in TxB₂ and bisenoic prostanoid production with no significant alterations in the proportion of TxB₂ production. Prostaglandin E₂ and prostaglandin F_2a, two prostanoids that can be formed either enzymatically or nonenzymatically from prostaglandin H₂, accounted for >75% of isolatable 20-carbon fatty acid cyclooxygenase products synthesized from endogenous and exogenous arachidonic acid.

¹ Research supported, in part, by the National Cancer Institute, Department of Health and Human Services, under Contract N01-CO-23910 with Program Resources, Inc.

² To whom requests for reprints should be addressed.

Received 11/ 2/87. Revised 2/ 2/88. Accepted 2/15/88.

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