| ObjectiveChronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western world, which is consist of 1/3 the leukemia, but much low frenquent in China. As the old people grow in number, the morbidity is increased. The DEK gene is at the 6p22.3, expressed in the proliferative cells and tumor cells. DEK was initially described as the target of a recurrent t(6;9) translocation in a subset of acute myeloid leukemia (AML) patients. Subsequently, it has been shown to promote tumorigenesis in a variety of cancer cell types, at least in part by its ability to interfere with cell division or DNA repair, inhibit cell differentiation, senescence, and apoptosis. Subsequent studies have repeatedly identified DEK as a frequently overexpressed gene in several neoplasms, included the CLL, and pro-oncogenic roles of DEK were supported by its ability to inhibit p53-mediated apoptosis. This study was aimed to investigate the expression level of DEK and its clinical features in CLL.MethodsmRNA levels of DEK genes were quantified using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with SYBR Green by ABI 7300 in 65 CLL patients. The relative expression levels of mRNA were analyzed by 2 (-ΔCT) method. Statistical analysis was performed by software SPSS (version 17.0). Mann-Whitney U-test determined differences of gene expression levels between different groups, and a P value of 0.05 was considered significant. Results①In 65 CLL patients, the median expression levels of DEK was 6.792×10-2 (1.438×10-2~3.201×10-1).②DEK expression level was not significantly correlated with the age (P=0.773), Binet stages (P=0.612), expression of serum lactate dehydrogenase (LDH) (P=0.115) andβ2-microglobulin (β2-MG) (P=0.330), and ZAP-70 expression (P=0.552). DEK expression level was significantly higher in patients with CD38-positive than in patients with CD38-negative (P=0.047). It was not found that there was correlated with the del(13q14) (P=0.159),+12 (P=0.546),IgH translocation (P=0.883) and del(11q22.3) (P=0.498), but it was significantly higher in patients with del(17p13) than in patients without del(17p13) (P=0.006) by Fluorescence in situ hybridization(FISH). It was not significantly correlated with the mutation of p53 gene (P=0.665). It was significantly correlated with the mutation of IgVH gene, the expression level was significantly higher in patients without Immunoglobulin heavy chain variable region (IgVH) mutation than in patients with IgVH mutation (P=0.025).ConclusionThe expression level of DEK were correlated with del(17p13) and the mutational states of IgVH gene, and it maybe prodicte the prognosis of CLL. ObjectiveChronic lymphocytic leukemia (CLL) is a heterogeneous malignant hematologic disease; the response to chemotherapy partly determines the prognosis of patients. Fludarabine, which is frequently used in the treatment of CLL, induces CLL cell apoptosis by activating p53, then the CLL cells into the mitochondrial apoptotic pathway and play a role in its anti-tumor. Nutlin-3 is potent and selective small-molecule antagonists of murine double mimute 2 (MDM2), and activate the p53 pathway in cells with wild-type p53. We incubated CLL cells with an active metabolite of fludarabine (F-ara-A) and Nutlin-3 in vitro, and then analyzed the role of DEK in p53-mediated apoptosis of CLL cells.MethodsCLL cells from 26 CLL patients were incubated in medium with 3.5 ?M fludarabine and 10 ?M Nutlin-3. Isometric cells were incubated with medium only, as blank control. CLL cells were harvested after 24-hour incubation. The apoptosis of cells was measured by staining with annexin V-FITC / propidium iodide (PI). The mRNA levels of DEK genes were quantified using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with SYBR Green by ABI LightCycler. Fluorescence in situ hybridization (FISH) analysis was used to detect ATM and p53 gene deletions. CLL cells were evaluated for immunoglobulin heavy chain variable region (IgVH) gene and p53 gene mutational status by PCR and sequencing. The function of p53 gene was detected by flow cytometry. Statistical analysis was performed by software SPSS (version 17.0). Differences of apoptosis levels and gene expression levels between CLL cells incubeted with or without fludarabine and Nutlin-3 were analyzed by matched-pairs t test. For all tests, a P value of 0.05 was considered significant.Results①The percentage of apoptosis at the beginning of incubation was 8.62% (1.83%~36.28%), and the percentages of spontaneous apoptosis after 24h incubation was 19.23% (7.96%~55.94%), and ?udarabine and Nutlin-3 induced apoptosis after 24h incubation were 40.85% (4.59%~75.31%) and 42.82% (18.11%~79.71%), respectively. When CLL cells were cultured in vitro, the spontaneous apoptosis was raised obviously (P=0.004). Median ?udarabine and Nutlin-3 induced apoptosis were significantly higher than the median spontaneous apoptosis of CLL cells (P<0.001, P<0.001, respectively).②In this analysis, the level of p21 in cytoplasm was not raised in CLL cells from 8 patients with p53 abnormality after incubated with fludarabine, 3 patients carried heterozygous ATM gene deletion, and elevation of p21 was detected in 2 CLL cells, after fludarabine induction.③After 24h incubation with fludarabine and Nutlin-3, compared to the control group, the expression of CLL cells with normal p53 function were significantly decreased (P=0.042, P=0.038, respectively), and the expression of CLL cells with abnormal p53 function were not changed (P=0.834, P=0.477, respectively).④After 24h incubation with fludarabine and Nutlin-3, compared to the control group, the expression of CLL cells without del(17p13) were significantly decreased (P=0.017, P=0.029, respectively), and the expression of CLL cells with del(17p13) were not changed (P=0.111, 0.378 respectively).⑤After 24h incubation with fludarabine and Nutlin-3, compared to the control group, the expression of CLL cells without p53 gene mutation were significantly decreased (P=0.037, P=0.017, respectively), and the expression of CLL cells with p53 gene mutation were not changed (P=0.263, P=0.378, respectively).ConclusionFludarabine and Nutlin-3 can be effective in inducing apoptosis in CLL cells. CLL cells with p53 gene deletion and/or p53 mutation showed dysfunction of p53. CLL cells with heterozygous ATM gene deletion carried functional p53. Down-regulating the expression of DEK depends on the functional p53. |
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