NK Cytotoxicity in Vitro and NK/ MDSCs/Tcell Subpopulations in AML/MDS Patients on 5-Azacytidine Treatment

5-Azacytidine is a cytosine analog and a potent DNA methyltransferase inhibitor, previously shown to induce DNA demethylation. 5-Azacytidine is indicated for the treatment of adult patients with intermediate-2 and high-risk myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) with <30 % blasts or of patients aged ≥65 years with AML who are not eligible for HSCT. NK cells are an important component of immunological tumor surveillance and their role in MDS pathogenesis is of rising interest. The role of immunosuppressive myeloid derived suppressor cells (MDSCs) in NK cell cytotoxicity and their correlation with lymphocyte subpopulations, is currently under investigation. With regard to MDS, increased NK-cell mediated cytotoxicity was found in one study, while several other studies reported impaired NK cell function ( Chamuleau ME et al. Hematologica 2009;94(4):496-50, Kiladjian JJ et al. Leukemia 2006;20(3):463-470, Epling-Burnette PK et al., Blood 2007;109(11):4816-482. We investigated 5-Azacytidine's impact on human in vitro NK cell cytotoxicity and in parallel its impact on frequencies of peripheral blood T, NKT, NK, Tregs and MDSCs cell subpopulations. To elucidate the immunological effects of 5-Azacytidine , we collected blood samples from 17 AML/MDS patients (age: 40-86y, median: 76y) and 9 healthy donors (age: 34-58y, median: 54y). Informed consent was obtained from all patients and donors according to the Declaration of Helsinki. Eleven patients suffered from MDS-RAEB II, three from secondary AML (MDS related) and three from AML. All patients had received at least 3 5-Azacytidine cycles (median 10, ranged from 3 -32). Complete remission was achieved by 47% of patients. Peripheral blood mononuclear cells (PBMC) were isolated from patients and healthy donors using density gradient centrifugation. CD56+/CD3- NK cells were purified from PBMCs by negative immunomagnetic selection. Purified NK cells were tested against the NK resistant Raji cell line and the NK sensitive K562 cell line, at a ratio of effector to target cells 5:1 . Cytotoxicity was measured in duplicate samples using a 4-h cytotoxicity assay at 37°C. Target cells were labeled with PKH-67stain and the analysis of cell viability was determined by staining with 7-AAD and was restricted to the PKH-67+ fraction. The mean proportion of 7AAD positive cells from the duplicate samples was determined. Background target cell death was determined from cells incubated in the absence of effector cells. Cell-mediated cytotoxicity was reported as the percentage of killing over background cell death averaged from the two samples. Frequencies of T, NKT, NK, Tregs and MDSCs cell populations were measured by flow cytopetry the same day as the cytotoxicity test was performed. MDSCs were phenotypically defined as CD33+/CD11b+/CD14-/HLA-DR lo/- . Statistical significance was determined by two-tailed unpaired t-test. Grouped data were expressed as mean ± standard error of the mean. The in vitro cytolytic capacity of AML/MDS-NK cells was investigated first against the erythroleukemia cell line K562, which represents a highly susceptible NK cell target and was presented significantly decreased compared with healthy donors (19,3±3,33 vs 38,3±5,68, p=0,0051)(graph 1.). In vitro cytotoxicity of AML/MDS-NK cells against NK resistant Raji cell line didn't show any difference between the two studied cohorts (patients:1,93±0,44 vs healthy donors:3,41±0,83, p=0,1). Overall frequencies of T, NKT, NK, TRegs and MDSCs cells were comparable among AML/MDS patients and healthy controls as it is presented in table 1, while CD3+/CD4+ subpopulation showed significant reduction in the patient's cohort. Natural Killer cell, well known to mediate anti-leukemic responses, showed defective in vitro cytoxicity in patient's cohort on 5'Azacytidine treatment , implying further in vivo impaired immune surveillance. Notably no reduced NK levels were found, nor any alterations in frequencies of MDSCs and Tregs were noted in patient's cohort. As it is already presented, MDS patients show a significant increase in MDSCs that negatively correlates with lymphocyte populations (Michelle K. Gleason et al.Blood, 2014; 123 (19):3016-3026). 5-Azacytidine treatment may confer to restitution of this impairment, conferring to meliorate antitumor immunity. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Cytotoxic Activity Of Non Polar Fraction From Annona Muricata L. Leaves On Hela And Raji Cell Line

<span>The main focus of the research is cancer by a viral infection such as nasopharinx and cervical cancer that have high incidence in Indonesia. Cervical cancer is due to the viral infection known as the Human Papilloma Virus (HPV) and nasopharing cancer is a cancer that is caused by EBV infection (Eppstein Barr virus). <span>The main priority of </span>nasopharinx<span> and servix cancer treatment is the use of chemotherapeutic agents, but it might also lead to diverse side-effects. The focus of this study was to develop the potency of non polar fraction from <em>Annona muricata</em> L. leaves by observing the cytotoxic effect on Hela and Raji cancer cell line.</span></span>

The Initial Research on Relationship of Transcription Factor C/EBPα and Raji Cell Line

Abstract 4650 Introduction: Leukemia is the common term for a diverse group of malignancies, defined by accumulation of abnormal hematopoietic progenitor cells, which fail to undergo terminal differentiation. The CCATT/enhancer binding protein alpha, C/EBPα, is a key transcription factor involved in normal hematopoietic system and leukemia. C/EBPα acts as an inhibitor of growth and apoptosis, and induces myeloid progenitors to differentiate. It is also reported that enforced expression of C/EBPα in B cells leads to their rapid and efficient reprogramming into macrophages. According to the phenomenon of cell-type transformation in clinical practice, we explore the role of C/EBPα in transformation of Raji cell line. Materials and Methods We detected expression of C/EBPα gene in Raji cell line by RT-PCR.The retroviral vector was inducted into the packaging cell Phoenix 293 by Lipofectamine™ 2000 (Invitrogen). Retroviruses encoding full-lengh cDNAs of murine C/EBPα were cloned by PCR into the BglII/XhoI sites of the pMIG retrovirus vector by creating a BglII site on the 5 end and a XhoI site at the 3 end. The supernatant of retrovirus was used to transfect Raji cells. To study the biological differences in C/EBPα positive-Raji cells, we used multiple methods such as cytomorphology, flow cytometry, MTT and RT-PCR. Results 1. Raji, 6T-CEM, Molt-4 and K562 cell line were negative for C/EBPα gene.However, HL60 and NB4 cells were positive for C/EBPα gene. Sequencing results showed that there is no genetic mutation. 2. The retrovirus pMIG and pMIG-C/EBPα were used to transfect Raji cell. The 72h transfection rates were 28.5% and 8.4% respectively. We used FACS to sort the GFP+ cells and amplification these cells. The GFP+ cells were taken account above 80%. By RT-PCR, we confirmed that C/EBPα gene was stably expressed. 3. Cytomorphology showed that the nuclein of Raji+pMIG-C/EBPα cells was loose compared to the other two cells. All the three cells were PAS and POX negative. FACS results showed that CD19 positive rate was (97.76±1.48)% A(97.93±0.64)% A(96.98±1.80)% in Raji cells, Raji+pMIG cells and Raji+pMIG-C/EBPα cells separately(P=0.688), and their mean value was 7003.5±276.48, 5803.5±159.10, 4808.5±327.39 (P=0.008). However, they were all negative for CD33 and CD14.RT-QPCR results showed that Raji cells, Raji+pMIG cells and Raji+pMIG-C/EBPα cells were Pax5 and PU.1 positive(P value was 0.450 and 0.186), and negative for MPO, G-CSFR and GM-CSFR gene. 4. We knew that C/EBPα gene may decrease proliferation of the cell. And this was maybe the reason for why we didn’t find a cell-type tansformation we expected. We did FACS 72h after transfection and found that CD19 positive rate of the Raji+pMIG-C/EBPα cell was decreased, but CD14 and CD33 were still nagetive. By sorting the CD19 negative cells which including all the GFP positive cells, we detected that apart from β-actin and C/EBPα gene were positive, the other five genes were all nagetive including Pax5 and PU.1. Conclusion C/EBPα acts as an inhibitor of growth in Raji cell line. B lymphoid cell related antigen CD19 and Pax5 and PU.1 genes were decreased after C/EBPα gene transfection.We conclude that more than one transcription factor were involved in this complex progress, and PU.1 may act an equal important role in it. Disclosures: No relevant conflicts of interest to declare.