Incorporation and effect of arachidonic acid on the growth of human myeloma cell lines.

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling-chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin A4 and B4, 12- and 15-hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.


Introduction
The lipoxygenase pathway converts arachidonic acid (AA), a 20-carbon polyunsaturated fatty acid, in hydroxyeicosatetraenoic acid (HETE), lipoxins (LX) and leukotrienes (LT). 1 Lipoxygenase inhibitors enhance the proliferation of human B cells in vitro, 2 while they have antiproliferative effects on malignant hematopoietic cell lines, 3,4 thus suggesting a role of endogenous lipoxygenase metabolites of AA during cell proliferation. While exogenous lipoxygenase products modulate cell proliferation in several cell types, 5 -7 their effect (if any) on the growth of human B cell lines are not documented. In this study we have investigated the incorporation of AA in three human myeloma cell lines (i.e. IM9, U266 and OPM2) and the effect of AA and lipoxygenase metabolites, such as LTB 4 , LTC 4 , LXA 4 , LXB 4 , 12-HETE and 15-HETE, on their proliferation.

Lipid extraction
Cells were mixed with 1 ml of 0.1% sodium dodecyl sulfate in water, and incubated for 30 min at 56°C with 3 ml of chloroform/methanol (2:1, v/v). The chloroformic extract was washed with 5 ml of 0.1 M KCl/ methanol/chloroform (96:92:6, v/v/v), and was evaporated. 8 The dry extract was recovered in 150 m l of chloroform and applied to a thin-layer chromatography (TLC) plate (Silica gel 60 (203 20 cm, 0.25 mm), Merck) and submitted to TLC. The plate was developed in the mixture of diethyl ether/hexane/acetic acid (70:30:1, v/v/v). 9 Each lane was divided into areas of 0.5 cm in length which were scraped into vials, and radioactivity was measured on a Packard liquid scintillation counter. Solutions of monoglycerides (MG), diglycerides (DG), triglycerides (TG), phospholipids (PL) and AA (Sigma, Saint Quentin Fallavier, France) were used as standards and visualized with iodine vapor.

Analysis of labeled PL
For the separation of the various species of cellular PL, the corresponding areas were scraped from the TLC plates and were extracted with chloroform/ methanol (2:1, v/v). Samples were then rechromatographed using a solvent system of chloroform/ methanol/acetic acid/water (50:30:8: 8 Each lane was divided into areas of 0.5 cm in length and processed as above. Solutions of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI) (Sigma) were used as standards and visualized with iodine vapor. In this chromatography, PI and PS migrated to the same area.

Base hydrolysis and PLA2 treatment of labeled PL
In a first set of experiments, the labeled PL were resuspended in 1 ml of 2 M KOH in ethanol/water (3:1, v/v) as previously reported. 9 After 40 min at 60°C, 1 ml of water and 1 ml of 6 N HCl were added to the mixture to acidify the phase (pH 3). Labeled compounds were extracted with chloroform/methanol (2:1, v/v) and rechromatographed using a solvent system of diethyl ether/hexane/acetic acid (70:30:1, v/v/v). The amount of radioactivity migrating with PL and free AA was determined by liquid scintillation counting. In another set of experiments, labeled PL were hydrolysed with PLA 2 from bovine pancreas (Sigma). Briefly, labeled PL samples were resus-

Results and Discussion
As reported in Fig. 1 and PL. Less than 10% of label is recovered in DG or as free AA, and labeled MG are not detected. A transfer of AA from TG to PL has been reported in several human cell types. 11  and IM9 cells (data not shown). Studies report that lipoxygenase and cyclooxygenase products of AA might derive from different PL species in some inflammatory cell types. 12 Thus, two sources of AA are released during immunological activation of mast cells. 14 PC provides AA for LTs biosynthesis, while PE provides the released AA that might be used for the production of cyclooxygenase products. Clearly the role of the different PL species on the production of lipoxygenase and cyclooxygenase metabolites of AA by human myeloma cells deserve to be investigated.
The addition of lipoxygenase metabolite of AA is reported to modulate the growth of various cell types. 5 -7 We then assessed their putative role on the proliferation of human myeloma cells. As reported in Table 2, the addition of micromolar amounts of AA, LTB 4 , LTC 4 , LXA 4 , LXB 4 , 12-HETE and 15-HETE has no significant (P>0.05, Mann-Whitney U-test, four independent experiments) effect on the [ 3 H]thymidine incorporation of U266, IM9 and OPM2 cells. This result is in accord with the fact that the enhanced B-cell proliferation caused by lipoxygenase blockade could not be reversed by the exogenous addition of LTs or HETEs. 2 In this study we have only investigated the effects of lipoxygenase products of AA on the growth of myeloma cells. However, cyclooxygenase products of AA such as prostaglandin E 2 are reported to modulate the proliferation of several cell types. 15,16 Clearly their role on the growth of U266, IM9 and OPM2 cells deserves to be clarified.
In conclusion, while human myeloma cells incorporate AA in their membrane phospholipids and triglycerides, exogenous AA and lipoxygenase products are not important modulators of their proliferation, a result that markedly differs to their reported endogenous role.