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Effects of Phosphatidylethanolamine and Phosphatidylcholine in Membrane Phospholipid on Binding of Phorbol Ester in Rat Mammary Carcinoma Cells¹

Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309

Mammalian cells in culture can be classified as either ethanolamine (Etn)-responsive or Etn-nonresponsive with regard to their growth. Epithelial cells and some of their transformed derivatives are the Etnresponsive type. When these cells are grown without Etn, the content of membrane phospholipid becomes significantly altered. Namely, the content of phosphatidylethanolamine is reduced and that of phosphatidylcholine is increased. In addition, the growth rate of these cells is reduced. Therefore, it is likely that the phosphatidylethanolamine deficiency or phosphatidylcholine excess is unsuitable for some membrane-associated functions resulting in the cessation of growth. In order to test the above hypothesis, we examined the binding of a tumor-promoting phorbol ester, [³H]phorbol 12,13-dibutyrate (PDB), to an Etn-responsive rat mammary carcinoma cell line 64-24 grown with (Etn-plus) or without Etn (Etnminus). The time course of binding was very similar between Etn-plus and -minus cells, except that the level of saturation was higher in Etn-plus cells, whereas the time course of chase of the bound PDB was significantly different between the two types of cells. Both types of cells have one class of binding sites for PDB. The dissociation constant (K_d) for [³H]PDB in Etn-plus cells was 34.0 nM and the number of binding sites at saturation was 2.7 x 10¹²/mg protein or 3.6 x 10⁵/cell. The corresponding values in Etn-minus cells were 61.4 nM and 3.2 x 10¹²/mg protein or 5.4 x 10⁵/cell, respectively. Although the difference in K_d values of the two types of cells was only 2-fold, this difference was statistically significant. On the other hand, the number of binding sites/mg protein in these cells was very similar. Since the amount of protein/cell was 1.4-fold higher in Etn-minus cells as compared to that of Etn-plus cells, the number of binding sites/cell was larger in Etn-minus cells. PDB affected the rate of proliferation of 64-24 cells differently, depending on whether they were grown in the presence or absence of Etn. These results suggest that the phosphatidylethanolamine and/or phosphatidylcholine content of the membrane phospholipid affects cellular functions mediated by phorbol esters.

¹ This work was supported by National Science Foundation grant PC8104480 and NIH Grant CA30545.

² To whom requests for reprints should be addressed.

Received 2/ 6/87. Revised 9/28/87. Revised 12/ 4/87. Accepted 12/ 9/87.