The Significance Of CD44ICD In Difuse Large B-cell Lymphoma Cell Line SUDHL2and The Preliminary Study On The Mechanism

BackgroundDiffuse large B cell lymphoma (the diffuse large B-cell lymphoma, DLBCL),is the one of the most common type of non-Hodhkin’s lymphoma, accounting for30%-40%of adult lymphoma. According to incomplete statistics, the incidence in China is higher accounting for about56%, and incidence rates are increasing every year. DLBCL is the disease of invasive and highly heterogeneous, and only about half of the patients with DLBCL achieved complete remission, treating with the standard CHOP or R-CHOP chemotherapy in clinical practice. So how to improve remission rate of refractory or relapsed DLBCL is the great challenge for the clinical doctor. IPI (International Prognostic Index IPI) is commonly used in clinical for prognostic indicators, but due to a high degree of heterogeneity of DLBCL, IPI score is difficult make accurate judgmentson the prognosis. By cDNA microarrays and oligonucleotides microarray techonology, contrasting the inactivated B cells, activated B cells in vitro and normal germinal center B-cell gene expression profiles, DLBCL is divided into three type:germinal center B cells (the germinal center B-cell-like, GCB) and activated B cells (activated-B-cell-like, ABC) and type3. The response to chemotherapy of GCB-type is much better than that of ABC-type, and the prognosis of the patients with GCB-type is better.The invasiveness and metastasis of tumor cells is closely related to adhesion molecules on their surface. CD44is a transmembrane adhesion molecule, widely expressed with the surface of normal cells and tumor cells, which involved in tumor cell differentiation, growth, and apoptosis regulation. Our previous study also find that CD44is closely related to the prognosis of DLBCL. The patients with CD44H or CD44v6positive have a poor prognosis than those without them. CD44are composed by four functional parts:signal peptide, the N-terminal extracellular domain, transmembrane domain and the C-terminal cytoplasmic domain, molecular size is about80-90KD. Transmembrane segment of the CD44molecule contains two progeria factor-dependent restriction sites near the cytoplasmic side of the hydrolyzed off, forming about12-16KD CD44intracellular domain (CD44ICD), and then enter the nucleus, activate the nuclear transcription factor NF-κB and other transcriptional regulation.Extracellular regulated protein kinase (ERK) includes ERK1and ERK2was referred to ERK1/2collectively, relative molecular weight was42KD and44KD,which are serine/threonine kinase driven by proline,leading to serine/threonine near proline phosphorylate.And ERK1/2is the one of mitogen-activated protein kinase’s(MAPK) family. After the phosphorylation, ERK1/2can go into the nucleus to promote the transcription of many transcription factors and the expression of certain genes. Excessive activation of ERK1/2is related to the tumor.The Notch receptor protein Notch gene encodes a transmembrane protein of the extracellular domain, transmembrane and intracellular segments. Mammals has four kinds of Notch receptors:Notch1-4. When the ligand and Notch receptor binding, protein hydrolysis reaction can happen twice. Wheny-secretase contact with its transmembrane region, the occurrence of the second hydrolysis reaction happens releasing of a free package of the Notch protein segment (NICD). NICD can go through the nuclear membrane into the nucleus, and bind with the transcription factor, regulate cell proliferation, differentiation, and apoptosis. It is reported in that down regulate the Notch-1receptor, NF-κB and its target gene can be inactivated, inhibiting tumor cell invasion and metastasis and inducing apoptosis.DAPT a synthetic γ-secretase inhibitor, currently recognized as the Notch receptor inhibitor, can effectively inhibit its hydrolysis, block its signaling pathway, thus affect cell proliferation, differentiation and apoptosis. DAPT also can inhibit CD44hydrolysis CD44ICD, thereby affecting the signaling pathway. Our previous experiments showed that when adding the10uM DAPT, the expression of CD44ICD and NF-κB was significantly affected suppressed. Meanwhile also DAPT enables the expression of CD44down-regulated. Therefore, we can not clear when treated the CD44-positive ABC-DLBCL cell lines, what caused the decline of NF-κB? Whether the decline of CD44ICD the expression resulting in NF-κB suppression or inhibition of Notch signal pathway decline the NF-κB expression, or both. ObjectiveUsing ABC-DLBCL cell lines, observe the location of CD44ICD by western blot and its influence on apoptosis. And explore the relationship among DAPT, the Notch signaling pathway, and CD44, make clear that which play the key role in the change of some protein after treating with DAPT, CD44or Notch signal pathway. Meanwhile observe the apoptosis after blocking CD44ICD, and provide some basic information and find ways to improve the response rate of DLBCL.Material and Method1. Cell lines:ABC-DLBCL cell lines:Sudhl2and OIC-ly3(from professor Ye BH, in the US);2. Test the expression of CD44:test the surface expression of CD44in SUDHL2、 OCI-ly3cell lines using flow cytometry;3. Deal with the wo cell lines with different concentration of DAPT, after24,48,72h test CD44expression and apoptosisthick with flow cytometry, and real-time PCR detect the gene change of Notch-1, CD44ICD, watch the morphology by microscope after Giemsa-Wright’s staining.4. Test whether ERK1/2and phosphor-ERK1/2expression:using western blot test to test them, and then deal with SUDHL2cell line with different concentration of DAPT for1h, test them by western blot angain;5. All data were statistically analyzed using SPSS13.0software. If the data is normally distributed, factotial design or One-Way ANOVA could be used, if the data showed non-normal distribution, non-parametric test must be used. Test level of α=0.05, two-sided test, P <0.05was statistically significant.P<0.05is accepted as an indication of statistical. Results represent the mean±D.Results1、The expression of CD44on two cell lines of DLBCL Flow cytometry tested Sudh12and OCI-Ly3cell lines, the expression of CD44were98.86%±1.56%,3.66%±3.72%respectively;2%The expression of Notch-1receptor in SUDHL2cell lines Sudhl2cell lines can be detected expression of Notch-1receptor by RT-PCR;3, The expression of CD44、CD19、CD20after treated with DAPT Treated SUDHL2cell line with0,0.1,1.0,5.0,10umol/L DAPT respectively for24h,48h after that test the expression of CD44by flow cytometry, the expression of CD44(F=0.371, P=0.826)、CD19(F=0.164, P=0.954)、CD20(F=0.049,P=0.995)by different concentration had no difference statistically; neither at differert time CD44(Z=-1.445, P=0.148)、CD19(Z=0.148, P=0.688)、CD20(F=2.494, P=0.130); there is no cross effect between concentration and time (F=0.371、0.164、0.049, P=0.826、0.954、0.995)。4、The effect of DAPT on CD44ICD and Notch-1gene expression Different concentrations of DAPT treated for24h,48h,72h respectively, CD44ICD mRNA and Notch-1mRNA expression was not significantly different (F=0.001、0.623, P=1.000、0.650) at different concentration, neither at different time point,(F=0.189,2.830, P=0.828,0.73); there is no cross effect between concentration and time(F=0.002,0.368,0.049, P=1,000、0.9265、The optopsis of SUDHL2cell after treated with DAPT Different concentration of DAPT displayed no difference (F=1.747, P=0.166); neither of the time point (χ2=1.249,P=0.539), and there is no cross effect between concentration and time (F=0.801, P=0.607)6, Expression of ERK1/2and phosphor-ERKl/2ERK1/2and phosphor-ERK1/2could be tested in SUDHL2cell line. When treated with different concentration of DAPT, the expression of ERK1/2and phosphor ERK1/2didn’t change;Conclusion1、 The γ-secretase inhibitor DAPT could not suppress the expression of CD44in SUDHL2cell lines, and had no effect on the expression of CD19, CD20, and no significant changes could be found in morphology2、 DAPT had no impact on the CD44ICD gene expression with different concentrations at the same point in SUDHL2cell line, and neither of the gene expression of Notch-13、 SUDHL2cell line could’t be induced to optoposis by low concentration of DAPT4、 SUDHL2cell lines from ABC-DLBCL sources expressed ERK1/2and phosphorylation of ERK1/2, inhibiting CD44ICD secretion with DAPT, couldn’t affect the expression of ERK1/2and phosphorylation-ERK1/2.