The intent of our original grant proposal was to utilize a very unusual invasive bacterial toxin, Bordetella Adenylate Cyclase Toxin (BACT), to assess the effects of cyclic AMP (cAMP) elevation upon growth, phenotypic expression, and malignant biologic behavior of normal and transformed cells. We have found: 1) BACT invades every mammalian cell tested except the human rbc, thereby establishing its wide applicability as a tool for manipulating cAMP; 2) only normal or relatively differentiated transformed cells undergo growth arrest and terminal differentiation in response to cAMP elevation, suggesting cAMP may be an endogenous regulator of normal cellular development; 3) BACT probably gains entry into target cells by binding to the sialic acid terminus of a ganglioside-like component; 4) cyclic AMP elevation in HL-60 leukemic cells causes a rapid and abrupt transcriptional downregulation of the c-myc oncogene that proceeds growth arrest and differentiation. We will examine the hypothesis that this downregulation reflects the functioning of a normal, """"""""physiologic"""""""" regulatory network that exerts its effect through cAMP-dependent protein kinase (CDPK). We will employ BACT and other cAMP-inducers to assess c-myc response to cAMP elevation in normal bone marrow hematopoietic precursors and in transformed, mostly hematologic cell lines with normal, amplified and rearranged c-myc genes. The pattern of response amongst cell lines with rearranged c-myc may divulge the location of a putative regulatory 5' sequences which confer cAMP-responsiveness to the c-myc gene. To determine how cAMP blocks c-myc transcription, we will employ inhibitors of other intracellular signaling networks, an isolated nuclear transcription runoff assay into which individual components of CDPK can be introduced, and a cAMP-resistant cell line. These studies should provide insights into the differential response of normal and transformed cells to cAMP elevation, the hitherto unexplored area of transcriptional downregulation of higher eukaryotic genes by cAMP, the role of CDPK in this process, and participation of the cAMP/CDPK system in differentiation caused by other, apparently unrelated agents such as phorbol esters, DMSO, and retinoic acid. These insights may, in turn, provide a rationale for attempting manipulation of cAMP levels in neoplastic cells with potentially targetable agents, such as BACT, as a therapy for cancer.
| Slungaard, A; Vercellotti, G M; Walker, G et al. (1990) Tumor necrosis factor alpha/cachectin stimulates eosinophil oxidant production and toxicity towards human endothelium. J Exp Med 171:2025-41 |
