Mdm4 Gene Expression And Clinical Significance In Chronic Lymphocytic Leukemia Research

ObjectiveHuman homolog of murine double minute4(MDM4) belongs to murine doubleminute (MDM) family. The gene is located at chromosome1q32and its cDNA codesfor a protein of490amino acids. Splicing variant of MDM4(S-MDM4) is obtainedfrom the deletion of exon6, which results in an internal deletion of68bp. Murinedouble minute2(MDM2) is an analogy of MDM4and shares highly similar structurewith each other. The MDM4gene plays a crucial role in regulating p53activity andhas been found to overexpress in chronic lymphocytic leukemia (CLL). The purposeof this study was to investigate prognostic significance of MDM4. Full-length MDM4(FL-MDM4), S-MDM4and murine double minute2(MDM2) mRNA expressionswere detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR) in140Chinese patients with CLL. This study was aimed to investigatethe expression level of MDM gene and its clinical features in CLL, and analyze therelationship of MDM gene and p53status.MethodsmRNA levels of FL-MDM4, S-MDM4and MDM2genes were quantified usingqRT-PCR with SYBR Green by ABI LightCycler in140CLL patients. The relativeexpression levels of mRNA were analyzed by2(-ΔCT)method. Statistical analysis wasperformed by software SPSS (version17.0). An effect was considered statisticallysignificant at P<0.05. Variables examined were age at diagnosis, gender, Binet stages,lactate dehydrogenase (LDH), thymidine kinase1(TK1), β2-microglobulin (β2-MG),immunoglobulin heavy chain variable region (IGHV) mutational status, molecularcytogenetic aberrations, p53mutations,CD38and ZAP-70protein. The difference ofMDM mRNA expressions between groups with different prognostic factors was described using the Mann–Whitney U test.Results①Ninety-six patients were male and44were female (male: female ratio,2.2), andthe median age was61years (rang:33–84). According to the Binet staging system,49(35%) patients were in stage A,53(37.9%) in stage B, and38(27.1%) in stage C.The qRT-PCR products were sequenced. The median expression levels of FL-MDM4,S-MDM4and MDM2were0.05274(0.1101–0.01342),0.01689(0.03349–0.00645)and0.01172(0.02717–0.00539), respectively.②p53aberrations were defined as del(17p13) and/or p53mutations. At the time ofenrollment, p53aberrations occurred in28of131CLL patients (21.4%).③FL-MDM4and S-MDM4expressions were significantly increased with thedel(17p13)(P=0.037and P=0.006), p53mutations (P=0.023and P<0.001) and p53aberrations (P=0.024and P<0.001). The correlation between the level of MDM2expression and p53status was not observed (P=0.196, P=0.095and P=0.092,respectively). Besides, a marked increase of S-MDM4expression was observed inCLL patients with advanced Binet stage (P=0.020), higher level of LDH (P=0.001)and β2-MG (P=0.026).④Survival analysis showed that high level of S-MDM4mRNA expression wasassociated with short treatment free survival (TFS). Del(17p13) was stronglyassociated with TFS by multivariate Cox regression analysis.ConclusionIn conclusion, high levels of FL-MDM4and S-MDM4mRNA indicate CLL patientswith p53aberrations. S-MDM4mRNA expression is correlated with a great deal ofclinical features and biocharacteristics, indicating a significant prognosis in CLL. ObjectiveChronic lymphocytic leukemia (CLL) is a heterogeneous malignant hematologicdisease; the response to chemotherapy partly determines the prognosis of patients.Fludarabine, which is frequently used in the treatment of CLL, induces CLL cellapoptosis by activating p53. The pharmacology of fludarabine is to inhibit DNAsynthesis, thus causing DNA damage and p53activation. Nutlin-3is a selectiveinhibitor for MDM2(murine double minute2)-p53interaction and rescues p53fromthe inhibition of MDM2, then leading activation of p53. FL-MDM4(full-lengthmurine double minute4), S-MDM4(splicing variant of MDM4) and MDM2containp53binding domain (BD) and exert their function through interaction with p53. Toidentify which is the key factor in p53pathway, we treatmented CLL cells withfludarabine or Nutin-3in vitro and then analyzed the relationship of apoptosis andp53status, and the role of FL-MDM4, S-MDM4and MDM2in p53-mediatedapoptosis of CLL cells.MethodsCLL cells from22CLL patients were treated with fludarabine or Nutin-3. Isometriccells were incubated with medium only, as blank control. CLL cells were harvestedafter24-hour incubation. The apoptosis of cells was measured by staining withannexin V-FITC/propidium iodide (PI). The mRNA levels of FL-MDM4, S-MDM4and MDM2were quantified using real-time quantitative reversetranscription-polymerase chain reaction (qRT-PCR) with SYBR Green by ABILightCycler. Fluorescence in situ hybridization (FISH) analysis was used to detect trisomy12,14q32translocation, and6q23,11q22.3,13q14,17p13deletion. CLLcells were evaluated for immunoglobulin heavy chain variable region (IGHV) geneand p53gene mutational status by PCR and sequencing. The function of p53genewas detected by flow cytometry. Statistical analysis was performed by software SPSS(version17.0). Differences of apoptosis levels and gene expression levels betweenCLL cells incubeted with or without fludarabine or Nutin-3were analyzed by t-pairstest. For all tests, a P value of0.05was considered significant.Results①The spontaneous apoptosis of CLL cells (n=22) were10.6%after24h incubation,which was significantly increased compared with that of at the beginning ofincubation (6.0%)(P=0.001). A markedly increase was observed in CLL cells treatedby fludarabine (n=22) or Nutlin-3(n=16) after24h incubation in vitro (P=0.001andP=0.001). p53aberrations were defined as del(17p13) and/or p53mutations. To studythe relationship of p53status and the level of apoptosis, we divided22CLL patientsinto two groups:13cases without p53aberrations and9cases with p53aberrations.Analyzing the13cases without p53aberrations, the apoptosis of CLL cells wasmarkedly increased after treatment with medium only or with24h incubation offludarabine or Nutlin-3(P<0.0001and P=0.006). However, the apoptosis was notsignificantly increased in CLL cells with p53aberrations.②In the primary CLL cells without p53aberrations or with p53normal function,FL-MDM4mRNA expression was significantly decreased after24h treatment withfludarabine (P=0.001and P=0.002), but increased after Nutlin-3treatment (P=0.008and P=0.011). A marked increase of S-MDM4expression was observed in the CLLcells after24h treatment with fludarabine (P=0.013and P=0.017). The level ofMDM2expression was elevated after24h incubation with fludarabine (P=0.030andP=0.007) and Nutin-3-treated (P=0.018and P=0.038).ConclusionFludarabine and Nutlin-3effectively induces apoptosis in CLL cells depended on functional p53gene. S-MDM4might be a more potent inhibitor of p53and probablythe drug target in the future.