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Effect of Nucleophosmin Mutation on Biologic Feature of Leukemia Cells and Potential Mechanisms

Author: LuoZhanPeng
Tutor: ZhangLing
School: Chongqing Medical University
Course: Clinical Laboratory Science
Keywords: Acute myeloid leukemia Nucleolar phosphoprotein Mutation Proliferation Apoptosis
CLC: R733.7
Type: Master's thesis
Year: 2008
Downloads: 97
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Abstract

Acute myeloid leukemia (acute myeloblastic leukemia, AML) is a hematopoietic malignancies, its rapid-onset, short course, a higher mortality rate. Traditional radiotherapy and chemotherapy for AML lower complete remission rate, easy to relapse. Although hematopoietic stem cell transplantation is the cure of AML, but there is rejection, the cost of treatment and many other issues. In recent years, found that leukemia related to the pathogenesis of a variety of fusion genes, these genes are relatively stable in the course of the disease, with the onset and progression of leukemia has important clinical significance. However, leukemia has a high degree of heterogeneity, a wide range of various types of leukemia-associated genes are closely associated with each type AML incidence has not yet found a specific gene. Recent studies have found that nucleolar phosphoprotein (nucleophosmin, NPM) gene mutations occur frequently in AML patients, and it can be found in the type AML M3 type. Clinical data showed that patients with AML in adults and children, NPM mutations have occurred, and its incidence increases with increasing age. Further study of NPM mutations usually involving leukemia stem / progenitor cells, and the involvement of the leukemia cells is often not associated with known AML fusion gene abnormalities, suggesting that NPM mutations may play a role in the early stages in AML. The clinical findings of NPM mutations is closely related to the prognosis of patients, not only can guide treatment choices and develop, but also can be used as a new indicator for monitoring minimal residual disease. Therefore, some scholars have suggested that the to the NPM mutations indicators included in the WHO classification standards of clinical AML, which further confirmed the important role of NPM mutations in clinical development, progress and outcome of AML. Recent discovery of NPM mutations promote normal mouse fibroblasts NIH3T3 malignant transformation, but also its impact on the biological characteristics of the leukemia cells have not been reported. Therefore, NPM mutations in the molecular mechanisms of the biological characteristics of the leukemia cells and is yet to be elucidated. This topic using gene transfection techniques, NPM mutations in the CD34 leukemia cell lines KG-1a for the study, analysis of mutant NPM and the molecular mechanisms of the early leukemia cell proliferation and apoptosis. The main results and conclusions are as follows: 1. Detect CD34 antigen KG-1a cells and mutant NPM gene expression. First detected by flow cytometry (FACS) CD34 cell content KG-1a cells, found that KG-1a cells CD34 cells accounted for 96.3%. Then were detected by RT-PCR and immunohistochemistry the mutant NPM gene expression and protein subcellular distribution confirmed KG-1a cells is the high expression of CD34 in the early leukemia cells, did not express mutant NPM gene and protein, in line with the follow-up experiments. 2 transfected with mutant NPM gene, to observe the impact on leukemia cell proliferation and apoptosis. First by liposome-mediated the the mutant NPM gene transfected into KG-1a cells and screened for stable expression strains. RT-PCR and immunohistochemical detection of the mutant NPM gene and protein expression, that mutant NPM gene and protein expression of leukemia cells after transfection; followed by FACS cell cycle, and found that the cell cycle progression transfection group was significantly faster, while Taiwan trypan blue staining to count living cells, cell proliferation ability; final spectrophotometric method to detect the activity of the cells casepase-3 to determine apoptosis, results showed that the transfected cells casepase-3 activity decreased, reduced apoptosis. The above experiments show the mutant NPM not only able to promote cell cycle progression and promoting cell proliferation in vitro, but also given the nature of the cells resistant to apoptosis. Preliminary molecular mechanisms explore the mutant NPM affect the biological characteristics of the leukemia cells. First of cell cycle regulators p21 mRNA levels, and found that the transfected cells, p21 mRNA expression reduce; then to detect cell casepase-9 activity was detected by RT-PCR, the transfected cells casepase-9 activity was significantly decreased. Confirmed transfected with mutant NPM can downregulate the expression of p21, promoting cell cycle progression, cell proliferation; Also by lowered mitochondrial apoptotic pathway regulating factor casepase-9 activity, causing apoptosis effector molecules casepase- 3 activity reduced, leading to cell resistance to apoptosis.

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