CAMP--ROLE IN GROWTH DIFFERENTIATION AND NEOPLASIA

我们最初的拨款提案的目的是利用一个非常 一种罕见的侵袭性细菌毒素--腺苷酸环化酶 毒素（BACT），以评估环磷酸腺苷（cAMP）的影响 生长、表型表达和恶性肿瘤后升高 正常和转化细胞的生物学行为。 我们有 发现：1）BACT侵入每一个哺乳动物细胞测试，除了 人红细胞，从而确立了其作为 操纵cAMP; 2）仅正常或相对分化 转化细胞经历生长停滞和终末 分化响应cAMP升高，表明cAMP 可能是正常细胞发育的内源性调节剂; 3)BACT可能通过与靶细胞结合而进入靶细胞。 神经节苷脂样成分的唾液酸末端; 4）环状 在HL-60白血病细胞中AMP的升高引起了快速和突然的 c-myc癌基因的转录下调， 进行生长停滞和分化。 我们会研究 假设这种下调反映了一种 正常的，“生理”调节网络发挥其作用 cAMP依赖性蛋白激酶（CDPK）。 我们会委聘 BACT和其他cAMP诱导剂，以评估c-myc对 正常骨髓造血前体细胞中cAMP升高 以及在转化的，主要是具有正常， 扩增并重排c-myc基因。 缓解模式 在c-myc重排的细胞系中， 假定的调节5'序列的位置，其赋予 cAMP对c-myc基因的反应性。 以确定如何 cAMP阻断c-myc转录，我们将采用抑制剂， 其他细胞内信号网络，一个孤立的核 转录径流测定法， 可以引入CDPK和cAMP抗性细胞系。 这些 研究应提供对不同反应的见解， 正常和转化细胞的cAMP升高，迄今为止， 转录下调的未探索区域 真核基因的cAMP，CDPK在这一过程中的作用， cAMP/CDPK系统参与分化， 通过其它的，明显无关的试剂，如佛波醇酯， DMSO和视黄酸。 这些见解反过来可以提供一个 试图操纵cAMP水平的基本原理 肿瘤细胞与潜在的靶向剂，如BACT， 作为癌症的治疗方法。

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.