| Background and ObjectivesAcute myeloid leukemia is a malignant and aggressive disease which occurs in hematopoietic stem and or progenitor cells. It is mainly characterized by that a large number of leukemia cells proliferate and accumulate rapidly in the bone marrow, resulting in inhibiting the function of normal hematopoietic cells. With a gradual upward trend about incidence and mortality rates, leukemia has been a malignant disease of hematopoietic system which threat human health seriously. In china, not only the incidence but also the mortality of leukemia rank top in malignant tumors.In the cancer registration area, the incidence and mortality of leukemia is 5.17/10 million and 3.49/10 million from 2003 to 2007, accounting for 1.94% and 2.29% of all malignant tumors, ranking No.13 and No.9, respectively. However, data analysis showed that the incidence and mortality of leukemia in our cancer registration area was 5.68/10 million and 4.28/10 million in 2009, accounting for 1.99% and 2.37% of all malignant tumors, showing an upward trend. With the reform and optimization of the current chemotherapy regimens,50 to 85% of patients can achieve complete remission (CR) after standard chemotherapy. Despite therapeutic improvements,30 to 40% AML patients suffer from disease relapse and progress to refractory leukemia, and even lead to death. Moreover, a portion of patients appear primary refractory to chemotherapeutic treatments even fail to achieve CR after standard induction program. In the final analysis, leukemic cells occur primary or secondary multidrug resistance to chemotherapeutic drugs is a major factor leading to the failure of chemotherapy and relapse. Drug-resistance continues to be a key obstacle for the successful treatment of AML.As drug resistance in the clinic, the processes of leukemia cells resistant to chemotherapy is also a complex processes of multiple factors involved and multi-step to complete. Recent studies showed that overexpression of drug-resistant gene such as MDR1, MRP1, BCRP, LRP, GSTP1, DHFR, gene mutation such as FLT3 and MLL, and the effect of bone marrow microenvironment, may contribute to drug-resistance of leukemic cells. Our previous study showed that in AML patients with AML1/ETO+, those with high expression of APP mRNA may have lower CR rate and poorer prognosis than the low expression group. Recently, scholars reported that the overexpression of EZH2 contributes to the acquired cisplatin resistance in ovarian and pancreatic cancer cells and 5-FU resistance in hepatocellular carcinoma cells. So, we have a hypothesis that, APP and EZH2 may contribute to chemotherapeutics resistance in AML1/ETO AML. But there is no related research about the relationship between the overexpression of APP and EZH2 gene and the chemotherapeutics resistance in AML cells. Meanwhile, our study has revealed that AML cell line Kasumi-1 overexpress APP and EZH2 and express AML1-ETO fusion gene. Furthermore, the migration ability of Kasumi-1 cells will degrade after silenting APP and EZH2 gene. On the basis of these findings, we set Kasumi-1cell line as the subject in this study, to detect its basic biological characteristics and the sensitivity to chemotherapy drugs, and to study the mechanism of APP and EZH2 gene participating in chemotherapeutic drug-resistance in Kasumi-1 cells, so as to providing a theoretical basis for finding targeted therapies to improve leukemia chemosensitivity.The AML1-ETO fusion transcription factor is generated by the translocation between chromosomes 8 and 21, which is the most frequent chromosomal abnormality in AML, occuring in approximately 4-12% of adult and 12-30% of pediatric acute myeloid leukemia patients. AML1-ETO positive leukemia exhibits some degree of myeloid differentiation, which led to their classification as the M2 subtype of AML, based on the French-American-British (FAB) Working Group Classification system. Both human and mouse models of AML have demonstrated that AML1-ETO can make contribution to leukemogenesis with the cooperation of other secondary events such as constitutively activated tyrosine kinase mutations, WT1 overexpression, ICSBP loss and p21 loss. Treatment for t(8;21) positive AML patients typically begins with conventional induction chemotherapy that based on cytarabine and an anthracycline (daunorubicin or idarubicin) (7+3). If a complete remission is achieved after the induction chemotherapy, high dose cytarabine is given as consolidation chemotherapy. Although AML patients harboring the t(8;21) translocation are generally given good prognosis and a majority of patients achieve complete remission, the 5-year survival is only about 50%,and the presence of a c-kit mutation can decrease the prognosis significantly.Our early clinical study showed that AML patients harboring the t(8;21) translocation and accompanying APP overexpression are given poor prognosis.It may reveal that APP overexpression is an unfavorable prognosis factor.Amyloid precursor protein (APP) is a type I transmembrane protein which widely express in various cell types. APP has many biological functions such as regulating cell growth and apoptosis, involved in gene transcription, adjusting calcium ion balance and so on. Some researches have demostrated that APP was over expressed in various solid tumors such as pancreatic cancer, oral squamous cell carcinoma, colon cancer, and it may promote the proliferation of cancer cells. We have reported that the expression of APP mRNA is the highest in AML1/ETO+AML among all kinds of AML, and the APP over-expression is related to migration and extramedullary infiltration of leukemia. But there is no report about the relationships between APP gene expression and chemosensitivity.The polycomb group protein enhancer of zeste homologue 2 (EZH2),which has histone methyltransferase activity, is a family member of PcG protein and the catalytic subunit of PRC2. EZH2 plays a transcription inhibitive function through methylation of H3K27 histone modifications and leads to tumorigenesis. Many malignancies, for example, prostate cancer and breast cancer, were found to over expressed of EZH2. Indepth studies have shown that EZH2 participate in the occurrence and evolution of the tumor through promoting tumor cell proliferation, migration and invasion, and arresting cell cycle. It is also reported that EZH2 over-expression is associated with poor prognosis and high aggressive in cancer. But currently there is no research on the role of EZH2 in the drug resistantce of leukemia cells.Kasumi-1, HL-60 and HL-60/ADM cell lines are myeloid cell lines, which are commonly used in scientific haematological research, however, the biological characteristics of these cell lines were researched rarely. Therefore, we set AML cell lines as the subject in this study. Firstly, cell morphology, flow cytometry immunophenotyping (FCM), karyotyping, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) technology and DNA sequencing methods were used to detect the basic biological characteristics of Kasumi-1, HL-60 and HL-60/ ADM cell lines. Then, qRT-PCR and Western Blot were used to test the expression of APP and EZH2 in the three cell lines. The drug sensitivity of the Kasumi-1, HL-60 and HL-60/ADM cell lines to Ara-c, HHT, ADM, DNR, IDA were detected by MTT assay. Furthermore, after using lentivirus-mediated gene transfer technique to silent the APP and EZH2 in Kasumi-1, the drug sensitivity to Ara-c and ADM were also detected by MTT assay. And the qRT-PCR was employed to evaluate MDR1 gene expression in Kasumi-1 before and after gene silenting. At last,in order to study how the APP and EZH2 gene involve in drug resistance, we used Western Blot to detect expression of Gli-l,a critical protien in Hedgehog pathway.Objicets and methods1. The biological characteristics of AML cell linesCell morphology, flow cytometry immunophenotyping (FCM), karyotyping, fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) technology and DNA sequencing methods were used to detect the basic biological characteristics of Kasumi-1, HL-60 and HL-60/ADM cell lines.qRT-PCR was used to detect APP and EZH2 mRNA expression levels in Kasumi-1, HL-60 and HL-60/ADM cell lines. Western Blot was used to detect APP and EZH2 protein expression levels in Kasumi-1, HL-60 and HL-60/ADM cell lines.2. Drug sensitivity of Kasumi-1, HL-60 and HL-60/ADM cell lines to comon chemotherapeutic drugsThe proliferation inhibition of the Ara-c,HHT,ADM,DNR,IDA to Kasumi-1, HL-60 and HL-60/ADM cell lines were detected by MTT assay, respectively. Then calculated the IC50 by an IC50 calculation software and calculated the resistant index(RI) by the formula IC50 (HL-60/ADM)/IC50 (HL-60),we can define that HL-60 is sensitive to the chemotherapeutic drugs and the HL-60 can be used as control cell line in the study when the RI exceeded 30.The ratio of IC50 (Kasumi-1) /IC50 (HL-60) was the RI of Kasumi-1 to chemotherapeutic drugs, we can define that Kasumi-1 is resistant to the drug if the RI exceeded 30.3. Reversion of drug-resistance to Ara-c and ADM after silencing APP or EZH2 gene in Kasumi-1 cellsAfter transfering a nontargeting scrambled into the lentivirus vector, our preliminary work have generated a negative control,which was used to transfect the Kasumi-1 cell to get the NC-Kasumi-1 as a blank control cell. Meanwhile,we have generated the si-APP Kasumi-l and si-EZH2 Kasumi-1 cells by transfecting Kasumi-1 cells with lentivirus vector that carried targeting scrambleds. The proliferation inhibition of the Ara-c and ADM to NC-Kasumi-l,si-APP Kasumi-1 and si-EZH2 Kasumi-l were detected by MTT assay. The difference of the proliferation inhibition between NC-Kasumi-1 and si-APP Kasumi-1, si-EZH2 Kasumi-1 should be compared.Then calculated the IC50 by an IC50 calculation software and calculated the reversal fold (RF) by the formula IC50 (Kasumi-1)/IC50 (si-Kasumi-1), if the RI exceeded 1, we can define that the drug resistance of Kasumi-1 cell was reverse after silenting APP and or EZH2 gene.4. Detected the signaling protein of drug-resistance reversal pathwayThe qRT-PCR was employed to evaluate MDR1 gene expression in Kasumi-1 before and after gene silenting. At last,in order to study how the APP and EZH2 gene involve in drug resistance, we used Western Blot to detect expression of Gli-1.5. Statistical analysisSPSS 13.0 software was used for the statistical analysis. Data were obtained from three independent experiments and expressed as means±standard deviation. Statistical analysis was performed by one-way ANOVA followed by the LSD test with homogeneity of variance or by the Dunnett’s T3 test with heterogeneity of variance for significance. p<0.05 is considered statistically significant.Results1. The comparision of biological characteristics of AML cell linesMorphology and cell phenotype detections showed Kasumi-1, HL-60 and HL-60/ADM cell lines all have the characteristics of myeloid leukemia cells. Karyotype analysis showed that three cell lines all possess complex chromosome karyotype and the Kasumi-1 cell accompanies with t(8;21). FISH and PCR test showed that Kasumi-1 cell line expresses AML1-ETO fusion gene. PCR and DNA sequencing detection showed that Kasumi-1 cells are positive for KIT gene mutation, but NPM1, FLT3-ITD and DNMT3AR882 mutation are negative. KIT, NPM1, FLT3-ITD and DNMT3AR882 mutation and the AML1-ETO fusion gene are negative in HL-60 and HL-60/ADM cell lines.2. Comparing the expression of APP and EZH2 in AML cell lines(1) A260/A280 values of all samples performed with qRT-PCR range from 1.80 to 2.00, suggests that RNA purity of samples meet the test requirements. And the melting curves of the target genes and the reference gene display a single peak, indicate that the melting temperature of cDNA were consistent, and the curve sharp peaks indicate high specificity primers. Superposition and parallelism of amplification curve showed that the experimental results are stable and reliable.(2) The expression levels of APP mRNA in HL-60, HL-60/ADM and Kasumi-1 are 0.142±0.049、0.053±0.009、0.171±0.065 respectively. Statistical analysis suggests that expression levels of APP gene in HL-60/ADM cells is significantly lower than Kasumi-1 and HL60 cells (P<0.05); but there were no significant difference between Kasumi-1 and HL60 cells(P=0.413).The expression levels of EZH2 mRNA in HL-60, HL-60/ADM and Kasumi-1 cells are 0.136±0.033, 0.121±0.019,0.114±0.015 respectively. Statistical analysis suggests that expression levels of EZH2 gene in HL-60, HL-60/ADM and Kasumi-1 cells have no significant difference (P>0.05).(3) Western Blot showed consistent internal control protein GAPDH, and the bands of internal reference protein and interest protein were clear, indicating that the experimental results are stable and reliable. Western Blot results show that the expression of APP was higher in Kasumi-1 cells than in HL-60/ADM cells, but there was no significant difference compared with HL-60, the expression of EZH2 also has no significant difference in the three cell lines.3. Drug sensitivity of Kasumi-1, HL-60 and HL-60/ADM cell lines to common chemotherapeutic drugsMTT assay showed that Ara-c, ADM, DNR, IDA, HHT inhibit the proliferation of HL-60, HL-60/ADM and Kasumi-1 cells in a dose-dependent manner.The proliferation inhibition rate increased with dose increase.The resistance index of HL-60/ADM cell line to Ara-c, ADM, DNR, IDA, HHT are exceed 30,indicating that HL-60/ADM cells are resistant to all the five chemotherapy drugs but the HL-60 cells are sensitive to the drugs.The resistance index of Kasumi-1 cell line to Ara-c, ADM, DNR, IDA, HHT is 1548,31,15,11,0.27 respectively, indicating that Kasumi-1 cells are resistant to Ara-c and ADM but sensitive to HH,DNR and 1DA. 4. Drug sensitivity of si-APP Kasumi-1 and si-EZH2 Kasumi-1 to Ara-c and ADMMTT assay showed that Ara-c and ADM inhibit the proliferation of NC-Kasumi-1, si-APP Kasumi-1 and si-EZH2 Kasumi-1 cells in a dose-dependent manner.The proliferation inhibition rate increased with dose increase. There was no significant difference in the proliferation inhibition of Ara-c and ADM between NC-Kasumi-1 and Kasumi-1 cells,indicating that virus transfection had no significant effect on cell proliferation.The reversal fold of si-APP Kasumi-1 cells to Ara-c and ADM were 1.4、0.28 respectively and the si-EZH2 Kasumi-1 cells were 59.35、64.03 respectively, showing that silenting the EZH2 gene can significantly increase the drug sensitivity in Kasumi-1 cells,but silenting the APP gene just increase the drug sensitivity in part.5. The mechanism of si-EZH2 Kasumi-1 reverse drug-resistanceResults of qRT-PCR showed that the expression of MDR1 mRNA in Kasumi-1 cells was significantly higher than the si-EZH2 Kasumi-1 cells (p=0.001), but there was no difference compared with si-APP Kasumi-1 cells (p=0.307). Comparing the expression of Gli-1,the si-EZH2 Kasumi-1 cells was significantly lower than the Kasumi-1 cells (p<0.001).ConclusionsKasumi-1, HL-60, HL-60/ADM cells express surface antigens of myeloid character and possess complex chromosome karyotype. Only Kasumi-1 cell accompanies with chromosome karyotype of t(8;21). NPM1, FLT3-ITD, DNMT3AR882 mutation in these three cell lines are negative but AML1-ETO fusion gene and KIT mutation are positive in Kasumi-1 cells. The expression of APPmRNA and protein in Kasumi-1 cells are significantly higher than those in HL-60/ADM cells, but there is no significant difference in HL-60.Tthe expression of EZH2mRNA and protein has no significant difference among three cell lines. In vitro, the chemosensitivity of Kasumi-1 cells was higher to HHT、DNR and IDA than Ara-c and ADM. Silenting the EZH2 gene can significantly increase the drug sensitivity in Kasumi-1 cells, accompany with lower expression of MRD1mRNA and the Hedgehog pathway protien Gli-1,but silenting the APP gene just increase the drug sensitivity in part,with no difference of the expression of MRD1mRNA. EZH2 inhibitor may be expected to become a targeted therapy in refractory and relapsing AML. |
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