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HL-60 Cells Can Be Made Copper Deficient by Incubating with Tetraethylenepentamine^1,2,3,

Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611

A system for studying copper deficiency was developed in a cell culture model. HL-60 cells were incubated with three chelators known to bind copper. One chelator, tetraethylenepentamine (TEPA), reduced cellular copper levels and the activities of two copper-requiring enzymes, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and cytochrome c oxidase. The specificity of the chelator was assessed by incubating cells with both copper and TEPA and, in other experiments, with zinc and TEPA. Copper levels, Cu/Zn-SOD activity and cytochrome c oxidase activity were restored to control values when copper and TEPA were added to cultures simultaneously, indicating the TEPA was responsible for reducing these aspects of copper metabolism. Incubating with both zinc and TEPA reduced copper levels relative to the control, but did not reduce Cu/Zn-SOD activity to the same extent as TEPA alone. The chelation of copper was a time-dependent process that was stable for at least 4 d. Cell growth and viability were not affected by TEPA. Respiratory burst activity, an indicator of differentiation, was not affected by TEPA, demonstrating that the reduction of Cu/Zn-SOD activity was due to copper chelation and not due to changes in Cu/Zn-SOD protein levels that occur during differentiation. Loss of copper, as well as a reduction of the activity of two copper-requiring enzymes, provides evidence that TEPA is a useful compound for creating a functional copper deficiency in cell culture.

KEY WORDS: • tetraethylenepentamine • HL-60 cells • cytochrome c oxidase • copper deficiency • Cu/Zn-superoxide dismutase

¹ Presented in part at the Annual Meeting of the Federation of American Societies for Experimental Biology, April 1992, Anaheim, CA [Layden, M. & Percival, S. (1992) Reduced copper levels in HL-60 cell culture by tetraethylenepentamine. FASEB J. 6: A1209 (abs. 1579)].

² Supported by grant number DK43518 from the National Institutes of Health and by the International Life Sciences Institute-Nutrition Foundation through a Future Leader Award and by funds provided by the Center for Nutritional Sciences, University of Florida.

³ Florida Agricultural Experiment Station Journal no. R02566.

⁴ To whom correspondence should be addressed.

Manuscript received 21 May 1992. Revision accepted 25 August 1992.