Overexpression Of Cyclin D2 In Human Chronic Myeloid Leukemia

Objective: Cyclin D2 was one of the members of a growing superfamily of D-type cyclins, proteins viewed as the most fundamental candidates for the positive regulators of the so called restriction point, a key cell-cycle checkpoint in Gl. Thanks to their central role in linking exogenous growth-regulating stimuli with cell cycle machinery, their deregulation was commonly involved in development of many types of cancer. Reports implicating cyclin D2 in oncogenesis are now emerging as well, including examples of CCND2 gene activations via gene amplification in colon cancer, retroviral integration in T cell leukemia, and induction by Epstein Barr virus in B-cell transformation. Current research in the field is to clarify whether cyclin D2 classifies as a proto-oncogene. CML is a malignant disease of the human hematopoietic stem cell that progress in a multistep fashion, namely the indolent chronic phase and a more aggressive and terminal blast crisis. Overwhelming evidences have indicated a role for the deregulated ABL protein tyrosine kinase (PTK) of the Philadelphia (Ph) chromosome characterized by the t(9;22)(q34; 11) translocation in the aetiology of CML. Been able to initiate excessive tyrosine phosphorylation of different cellular substrates in vivo and in vitro including the BCR-ABL protein itself, the deregulated tyrosine kinase activity of the BCR-ABL fusion protein compared to normal ABL has been established as the causative molecular event in CML and has been designed as the direct target for pharmacologic inhibition in the therapeutic approach to this disease. By the mutational analysis, people have come to a conclusion that the PTK activity is an absolute requirement for malignant transformation. However, the exact mechanism of the elevated tyrosine kinase activity of BCR/ABL in promoting proliferation and survival associated with the cell cycle regulation has not been established. Our work is based on the primary human hematopoietic progenitor cell model of CML developed by the transduction of b3a2 BCR/ABL cDNA in normal CD34(+) cells and a MSCV-ib-IRES-eGFP retroviral vector constructed to express intracellular single-chain antibody (sFv/intrabody) directed against ABL tyrosine kinase domain. An in vitro cell model named K562-ib-eGFP expressing intracellular single-chain antibody (sFv/intrabody) directed against ABL tyrosine kinase domain was developed by transducing the MSCV-ib-IRES-eGFP into K562 cells. The relationship between the expression of cyclin D2 and the deregulatedPTK activity of p210BCR/ABL fusion protein was investigated using the two models.Methods: RT-PCR was performed to detect the mRNA expression of cyclin D2 using the model of human p210(BCR/ABL)-mediated CML model and the mock-transduced CD34(+) cells model as we have described previously. The MSCV-ib-IRES-eGFP and MSCV-IRES-eGFP plasmids were cotransfected with the pCL-Ampho packaging plasmid into 293kj cells using a modification of the HEPES-buffered saline calcium-phosphate method. The virus-containing supernatant was harvested on days 1 and 2 by removing medium with a syringe and filter through a cellulose-acetate 0.45 micron filter. Then, the K562-ib-eGFP cell model expressing intracellular single-chain antibody against the tyrosine kinase domain of BCR/ABL and c-ABL protein was constructed by transducting the BCR/ABL+ K562 cells with the MSCV-ib-IRES-eGFP virus-containing supernatant. The control was constructed the same way using the MSCV-IRES-eGFP retroviral vestor. The eGFP positive K562 cells were selected by FACS. PCR was performed to detect the expression of the single-chain antibody. Western blot was performed to detect the expression of BCR-ABL and c-ABL protein using a mouse abl antibody in K562, K562-ib-eGFP and K562-eGFP cells. To assess PTKactivity in the cells, we performed phosphorylation assays using a synthetic tyrosine-contaming peptide, RR-SRC, which is based on the sequence surrounding the site of autophosphorylation in pp60scc34. Then, RT-PCR, western blot and flow cytometry assays wer...