[CANCER RESEARCH 43, 4552-4556, October 1983] Antagonist of Phorbol Ester Receptor-mediated Chemotaxis in Mouse Peritoneal Macrophages1 Robert J. Sturm,2 Bonita M. Smith,3 Richard W. Lane,3 Debra L. Laskin,4 Louis S. Harris, and Richard A. Carchman5 Department of Pharmacology and Toxicology, Medical College of Virginia, Richmond, Virginia 23298 ABSTRACT Biologically active phorbol ester derivatives displace [3H]phor- bol-12,13-dibutyrate from thioglycollate-elicited mouse peritoneal macrophages in a time-, temperature-, and concentration-de pendent manner. Scatchard analysis revealed an apparent K,, of 54.1 nw and 8.0 x 10s sites/cell, indicating that these macro phages possess saturable, high-affinity phorbol ester-binding sites. These derivatives also act as chemoattractants for the macrophage at equivalent concentrations. A notable exception to this pattern is phorbol-12,13-diacetate. Phorbol-12,13-diace- tate inhibits specific binding of [3H]phorbol-12,13-dibutyrate (con centration required for a 50% inhibition of the maximum specific binding of [3H]phorbol-12,13-dibutyrate, 2.6 MM)and Chemotaxis to phorbol-12-myristate, 13-acetate (concentration required for a 50% inhibition of the maximum chemotactic response, 0.39 pu) while exhibiting no activity as a chemoattractant at concen trations up to 10~5 M. The data indicate that phorbol-12,13- diacetate may be an antagonist for receptor-mediated chemo- taxis to phorbol-12-myristate, 13-acetate in the macrophage. INTRODUCTION The reported biological responses altered by phorbol ester tumor promoters both in vivo (38) and in vitro (6,7) are extensive. This laboratory and others have been studying the effects of these compounds on certain macrophage functions. Such inves tigations have described facets of the phorbol ester-macrophage response with respect to phagocytosis (23), Chemotaxis (21), macrophage-mediated tumoricidal activity (18, 22, 27-29, 34), and lysosomal enzyme release (9). These effects occur at low concentrations and with structure-activity relationships compa rable to those obtained for tumor promotion in the mouse skin model (19). This information led us to surmise that some of these macrophage responses were receptor mediated. Recently, several laboratories have reported the presence of a specific, high-affinity phorbol ester receptor on a variety of cell types (13, 31, 35). Using pHJPDBU6 as the radioligand, this Received September 8,1982; accepted July 1,1983. 1Supported by grants from the American Cancer Society (IN-105G), the National Institute of Dental Research (5P50DE05139) and the United States Environmental Protection Agency (R-808861010). 2 Postdoctoral trainee supported by National Institute of Environmental Health Services Training Grant T32E507087. To whom requests for reprints should be addressed. 3 Predoctoral trainees supported by National Institute of Environmental Health Services Training Grant T32E507087. 4 Present address: The Wistar Institute of Anatomy and Biology, Philadelphia, Pa. 19104. 5 Recipient of NIH Research Career Development Award K04AM00565. 'The abbreviations used are: PDBU, phorbol-12,13-dibutyrate; PDA, phorbol- 12,13-diacetate; DMEM, Dulbecco's minimal esential medium; PMA, phorbol-12- receptor binds biologically active phorbol esters and related derivatives. The presence of such a binding site may help to explain how these compounds evoke such pleiotypic responses. Though the above findings represent an important advance in our knowledge of tumor promoters, the lack of a competitive antagonist for phorbol ester binding and function has been an impediment towards the further elucidation of those specific biological effects which are receptor mediated. The ability to isolate receptor occupancy from biological events is the basis for the use of an antagonist, since it allows for the separation of these events. The usefulness of such an antagonist is illustrated by analogous situations which exist in pharmacology. Compounds such as retinoids (42), nonsteroidal and steroidal antiinflammatory agents (3,4,36,44), and certain prostaglandins (4, 17, 44) are known to be functional antagonists of tumor promotion but do not interact with this receptor (35). Alterna tively, phorbol ester derivatives which are biologically inactive are also inactive as competitors for the PDBU binding site (13, 31,35). Existing literature indicates that highest biological activity requires esterified alkyl side chains with a combined length of 14 to 20 carbon atoms at these 2 positions (11). This activity falls off with any further increase or decrease in chain length. The derivative PDA, with a total of 4 carbon atoms at positions 12 and 13, has been reported to be inactive as a tumor promoter (2, 37), except when tested in a sensitive strain of mouse (1). The significance of PDA as an active tumor promoter may be of limited value since, in this same study, even the typically inactive parent alcohol, phorbol, demonstrated tumor-promoting activity. Alternatively, Blumberg et al. (8) suggest that these studies failed to use PDA at sufficiently high doses to demonstrate activity. We have examined several phorbol ester derivatives with various substitutions at positions 12 and 13 of the molecule for the purpose of detecting a compound with antagonistic proper ties. Preliminary findings from this laboratory indicated that PDA could effectively block Chemotaxis (i.e., directed movement of cells along the concentration gradient of a specific chemoattrac tant) to active phorbol esters while showing no activity as a chemoattractant itself. Consequently, we initiated studies to examine this molecule as a phorbol ester antagonist. Part of this work was reported previously in preliminary form (43). MATERIALS AND METHODS Materials. Female ICR mice were purchased from Dominion Labora tories (Dublin, Va.). Tissue culture reagents and media were obtained from Grand Island Biological Co. (Grand Island, N. Y.). All diterpene derivatives for this study were purchased from P-L Biochemicals, Inc. (Milwaukee, Wis.). These derivatives were resuspended to 10"2 M stock myristate, 13-acetate; POD. phorbol-12,13-dktecanoate; 1C»,concentration re quired for a 50% inhibition of the maximum response. 4552 CANCER RESEARCH VOL. 43 Research. on October 10, 2021. © 1983 American Association for Cancer cancerres.aacrjournals.org Downloaded from