MOLECULAR INHIBITION OF BCR-ABL TYROSINE KINASE BY BCR SEQUENCES

Bcr-Abl癌蛋白是费城的主要致病因素 染色体(Ph)相关性白血病。活化的酪氨酸激酶 Bcr-Abl癌蛋白是其致癌的主要推动力 活动。我们已经证明了删除形式的bcr[bcr(-413)]， 包围了BCR的Abl SH2结合结构域，降低了 细胞内c-Abl和bcr-Abl的磷酸酪氨酸含量，并受到抑制 Bcr-A体外自磷酸化活性。同样，BCR多肽 丝氨酸354磷酸化对c-Abl和bcr-Abl激酶的体外阻断作用 而未磷酸化的多肽没有抑制作用。BCR(-413)为 也抵抗活化的c-Abl或酪氨酸磷酸化 Bcr-abl.重要的是，bcr(-413)对bcr-abl细胞的生长有干扰作用 表达细胞衬里。我们的发现表明Abl SH2结合 磷酸丝氨酸形式的bcr结构域抑制bcr-Abl癌蛋白， 但BCR这一区域的酪氨酸磷酸化逆转了它的 Bcr-Abl的抑制作用。这些发现的结果形成了 为治疗慢性粒细胞白血病慢性期患者的新策略奠定了基础。具体的 目的是：1)确定BCR(-413)的最佳试验形式 表达bcr-Abl基因的人造血细胞逆转能力的研究 Bcr-Abl诱导的生长/凋亡效应。这些构造将是 与仅载体相比，bcr(1-413)和S354A bcr(-413) 细胞。这些研究将使用转基因和阳性 用四环素抑制因子启动子系统进行筛选。2)构建一个 重组复制缺陷型腺病毒Bcr(-413)用于插入 将bcr抑制物序列分为bcr-Abl阳性和阴性细胞系。 3)检测重组缺陷型bcr重组腺病毒在 用CML来源的细胞系编程的SCID/NOD SCID小鼠系统 病人。我们计划测试BCR(-413)对患者细胞株的作用 在SCID小鼠模型中生长。我们计划感染K562细胞和KBM-5 用腺病毒载体Ad5CMVBCR(-413)检测其对细胞生长的影响 这些患者细胞系在适当的SCID模型中的特性 与编码无关基因(抗病毒)的腺病毒制剂相比 正义C-CAM)。最近的研究结果表明，Ad5CMV BCR(-413)诱导了 K562细胞感染2天后，75%的细胞死亡。细胞杀戮 由于Ad5 CMV反义C CAM对BCR序列有特异性作用，因此对BCR序列有特异性影响 不影响细胞生长或诱导细胞死亡。4)测试的效果 重组复制缺陷型bcr(-413)腺病毒感染 慢性粒细胞白血病慢性期患者的人骨髓标本，Ph- ALL阳性，骨髓正常。这些影响将与 阴性对照：腺病毒。细胞死亡(50%)是在低水平诱导的 未经治疗的慢性粒细胞白血病患者的高密度骨髓制备。我们期待着 Bcr(-413)对Ph阳性克隆的生长有抑制作用。5) 研究人参皂苷合成肽的抑制作用 Bcr(-413)对bcr-Abl表达细胞的抑制作用 线条和骨髓样本。病毒介导的阳性结果中的一种 BCR(-413)或该BCR区的可溶性多肽的引入 将有助于包括在治疗方案中的治疗 慢性期CML患者。

The Bcr-Abl oncoprotein is the primary causative factor in Philadelphia chromosome (Ph) associated leukemias. The activated tyrosine kinase of the Bcr-Abl oncoprotein is the primary driving force behind its oncogenic activity. We have shown that a deleted form of Bcr [Bcr(64-413)], encompassing the Abl SH2 binding domains of Bcr, reduced the phosphotyrosine content of c-Abl and Bcr-Abl within cells, and inhibited Bcr-A autophosphorylation activity in vitro. Similarly, a Bcr peptide phosphorylated on Ser 354 blocked the c-Abl and Bcr-Abl kinases in vitro whereas the unphosphorylated peptide was not inhibitory. Bcr(64-413) was also resistant to tyrosine phosphorylation by either activated c-Abl or Bcr-Abl. Importantly, Bcr(64-413) interfered with the growth of BCR-Abl expressing cell liners. Our findings indicate that the Abl SH2 binding domain of Bcr in the phospho-serine form inhibits the Bcr-Abl oncoprotein, but that tyrosine phosphorylation of this domain of Bcr reverse its inhibitory effects of Bcr-Abl. The results of these findings form the basis of a new strategy to treat chronic phase CML patients. The specific aims are: 1) Determine the optimum form of Bcr(64-413) will be tested in human hemopoietic cells expressing Bcr-Abl for their ability to reverse growth/apoptosis effects induced by Bcr-Abl. These constructs will be compared to vector only, BCR(1-413), and S354A BCR(64-413) transfected cells. These studies will be done using transfection and positive selection with the tetracycline repressor promoter system. 2) Construct a BCR(64-413) recombinant, replication-defective adenovirus for insertion of the BCR inhibitor sequence into Bcr-Abl positive and negative cell lines. 3) Test the effectiveness of a recombinant defective Bcr adenovirus in SCID/NOD SCID mouse systems programmed with cell lines derived from CML patients. We plan to test the effects of Bcr(64-413) on patient cell lines growing in a SCID mouse model. We plan to infect K562 cells and KBM-5 cells with Ad5 CMV BCR(64-413) to determine its effects on the growth properties of these patient cell lines in the appropriate SCID model compared to an adenovirus preparation encoding a unrelated gene (anti- sense C-CAM). Recent findings indicate that Ad5 CMV BCR(64-413) induced cell death in 75% in K562 cells infected for 2 days. The cell killing effects was specific to BCR sequences because Ad5 CMV anti-sense C CAM did not affect cell growth or induce cell death. 4) Test the effects of recombinant, replication defective BCR(64-413) adenovirus infection in human bone marrow preparations from CML chronic phase patients, Ph- positive ALL, and normal bone marrow. These effects will be compared to negative control adenoviruses. Cell death (50%) has been induced in a low density marrow preparation from an untreated CML patient. We anticipate that Bcr(64-413) will inhibit growth of Ph-positive colonies. 5) Investigate the inhibitory effects of synthetic peptides derived from Bcr(64-413) for their utility as inhibitors of Bcr-Abl expressing cell lines and marrow samples. Positive results from either virus-mediated introduction of Bcr(64-413) or soluble peptides from this region of Bcr will be useful for inclusion in therapeutic protocols for treatment of chronic phase CML patients.