Objective: To investigate the possibility of multi-unit ribozymes to purge bone marrow of chronic myelocytic leukemia (CML), its in vitro cleavage ability and the reversal effect on CML cell's malignant phenotype.
Methods: As bcr-abl fusion gene plays an important role in CML pathology, three single-unit ribozymes were designed and synthesized in 44 base pairs near the fusion point, two enzyme cleavage sites on bcr gene and one on abl gene. Multi-unit ribozymes' in vitro transcription and retroviral vector through gene recombination were constructed. Then, its in vitro cleavage ability was tested and the retroviral vector was transfected into K562 cell. Through MTT assay, the incorporation rate of (3)H-TdR, RT-PCR, Southern and Northern blot hybridization, flow cytometry, transmission and scanning electron microscopy were used to study the effect of multi-unit ribozymes on CML cell proliferation, cell structure, cell cycle and the induction of apoptosis.
Results: Multi-unit ribozymes had in vitro cleavage efficiency of 70.8%. After the transfection of multi-unit ribozymes retroviral vector into K562 cell, cell proliferation and DNA synthesis were greatly reduced with an inhibition rate of about 50% after 96 hours of transfection. Multi-unit ribozymes could cleave K562 cell's RNA with a reduction rate about one 1 000 th of the original. By flow cytometry (FCM), 18.4% cells underwent apoptosis after 72 hours transfection with most of the cells blocked in the G phase. Here, the ratio in S phase was lowered by 41.9%. Under transmission and scanning electron microscope, compaction of nuclear chromation and apoptosis bodies were observed in the transfected cells.
Conclusion: Multi-unit ribozymes possess high cleavage ability in vitro. The ribozymes, whose retroviral vector being transfected into CML cell, are able to express a lasting ability to cleave the fusion gene, induce apoptosis, reduce cell proliferation, revert the malignant phenotype. It is possible to make use of multi-unit ribozymes to purge CML bone marrow. Therefore, multi-unit ribozymes may very well be valuable in the gene therapy of CML.