Snyder D S, Wu Y, McMahon R, Yu L, Rossi J J, Forman S J
Department of Hematology and Bone Marrow Transplantation, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA, USA.
Biol Blood Marrow Transplant. 1997 Oct;3(4):179-86.
The bcr-abl oncogene is the molecular counterpart of the Philadelphia chromosome (Ph), which is detected in > 95% of patients with chronic myelogenous leukemia (CML) and 20-30% of adults with acute lymphoblastic leukemia (ALL). Leukemic cells from patients with CML express the p210 form of the bcr-abl oncogene, whereas in adult Ph+ ALL approximately 50% of cases express the p190 form of the bcr-abl oncogene, and the other 50% express the same p210 gene as is found in CML. In this study, we have designed hairpin ribozymes (RZs) specific for the p190 form of the bcr-abl oncogene to inhibit the growth of a p190 Ph+ ALL cell line, Sup-B15. The RZs cleave p190 RNA substrate in a cell-free in vitro assay. In the presence of the liposome, DMRIE-C, the RZs are protected from serum mediated catalysis in vitro. Anti-p190 RZs transfected with DMRIE-C as the vector into K562 cells, which express the p210 bcr-abl oncogene, are stable intracellularly for up to 96 hours. Up to 33% of the DMRIE-C and RZ mixtures are taken up by Sup-B15 cells cultured in suspension. Expression of the p190 bcr-abl protein product is specifically inhibited as demonstrated by Western blot analysis. Cell growth of the Sup-B15 cells is completely inhibited by anti-p190 RZs over four days in culture. Anti-p210 RZs have no significant effect on bcr-abl protein expression or cell growth by Sup-B15 cells. RZs may have a role in purging stem cell populations collected from patients with Ph+ ALL in the context of autologous bone marrow transplantation.
bcr-abl癌基因是费城染色体（Ph）的分子对应物，在超过95%的慢性粒细胞白血病（CML）患者以及20%-30%的成人急性淋巴细胞白血病（ALL）患者中可检测到。CML患者的白血病细胞表达bcr-abl癌基因的p210形式，而在成人Ph+ ALL中，约50%的病例表达bcr-abl癌基因的p190形式，另外50%表达与CML中相同的p210基因。在本研究中，我们设计了针对bcr-abl癌基因p190形式的发夹状核酶（RZ），以抑制p190 Ph+ ALL细胞系Sup-B15的生长。这些RZ在无细胞体外试验中可切割p190 RNA底物。在脂质体DMRIE-C存在的情况下，RZ在体外可免受血清介导的催化作用。以DMRIE-C为载体转染的抗p190 RZ进入表达p210 bcr-abl癌基因的K562细胞后，在细胞内可稳定存在长达96小时。悬浮培养的Sup-B15细胞可摄取高达33%的DMRIE-C与RZ混合物。蛋白质印迹分析表明，p190 bcr-abl蛋白产物的表达受到特异性抑制。在培养四天的时间里，抗p190 RZ可完全抑制Sup-B15细胞的生长。抗p210 RZ对Sup-B15细胞的bcr-abl蛋白表达或细胞生长无显著影响。在自体骨髓移植的背景下，RZ可能在清除从Ph+ ALL患者采集的干细胞群体方面发挥作用。
