1.Study On The Cell-markers Of Leukemia Stem Cells 2.Effect Of G-rich Oligonucleotide On Leukemia Cells And Its Relationship With P53 Expression

Objective:Leukemia stem cell (LSC) is the main cause of leukemogenesis, drug-resistance and relapse of leukemia. They have the potential of self-renewal. It has been recognized that LSCs reside within the CD34+/CD38-/CD123+ compartment. However, the pure population of LSCs in patients with AML has not been identified. The interaction mediated by adhesion molecules between LSCs and BM stromal cells played a role in the disturbed self-renewing and inhibited apoptosis of LSCs, which may be one of the mechanism of escape from chemotherapy. Therefore, to study the cell surface makers related to niche on LSCs is helpful for the potential target therapy in the future. In this study, expression of the three adhesion molecules, the N-cadherin, Tie2 and CD44 in the proportion of CD34+/CD38-/CD123+ LSCs from AML patients before and after chemotherapy were analyzed. Whether the N-cadherin, Tie2 and CD44 could be the potential markers for identification of LSCs were investigated.Methods:Bone marrow mononuclear cells (BMMNCs) from the 63 AML patients were obtained at different time points including the day before chemotherapy (at time of diagnosis, Pre), one day after the induction chemotherapy (Post-Day1), seven days after the induction chemotherapy (Post-Day7) and twenty one days after the induction chemotherapy (Post-Day 21), and analyzed by fluorescence activated cell sorting (FACS) for the expression of N-cadherin, Tie2, CD44, CD34, CD38 and CD123. And the proportions of N-cadherin, Tie2 and CD44 in LSCs (CD34+/CD38-/CD123+ cells compartment) were investigated. Combination with immunophenotyping and fluorescence in situ hybridization (FISH) analysis, N-cadherin and Tie2 positive CD34+/CD38-/CD123+ LSCs proportions of BM samples derived from 4 AML-M2 patients were isolated by FACS sorting for expression of AML1/ETO. N-cadherin and Tie2 positive CD34+/CD38-/CD123+ LSCs proportions of 5 AML patients were isolated by FACS sorting for expression of MDR1 mRNA by real-time PCR.Results:①In BMMNCs of AML patients, no obvious increase in the proportions of LSCs is occurred at Post-Day 1. With the subsequent effect of chemotherapy and the complete elimination of chemosensitive cells, the proportions of N-cadherin and Tie2 positive CD34+/CD38-/CD123+ LSCs increased from 0.17% and 0.02% to 0.6% and 0.94% at Post-Day 7, respectively (P<0.05, P<0.01), but the proportions of CD44 positive CD34+/CD38-/CD123+ LSCs decreased from 17.28% to 2.25%(P<0.05) (median);②In CD34+/CD38-/CD123+ LSCs, the fold of increase in the proportions of N-Cadherin, Tie2 and CD44 positive in CD34+/CD38-/CD123+ LSCs populations at Post-Day 7 could be clearly demonstrated as 38.17 (P=0.01),210 (P=0.03) and 1.01 (P=0.15), respectively. Results further illustrated that CD34+/CD38-/CD123+/N-Cadherin+ and CD34+/CD38-/CD123+/Tie2+ cells compartments were more resistant to chemotherapy than CD34+/CD38-/CD123+/CD44+ cells compartment, and the former two cells compartments have the properties of LSCs, they may be the true LSCs;③The enrichment capability of N-cadherin and Tie2 positive LSCs compartment were stronger than CD44 positive LSCs compartment, which indicated N-cadherin and Tie2 positive LSCs compartment may be the true LSCs;④In patients did not achieve CR (non-remission, NR) group, the proportions of CD34+/CD38-/CD123+/N-Cadherin+, CD34+/CD38-/CD123+/Tie2+ and CD34+/CD38-/CD123+/CD44+ cells compartments in BMMNCs showed a tendency to be higher than that in complete remission (CR) group at the time of diagnosis (P>0.05), which proposed that N-Cadherin, Tie2 and CD44 expression may serve as markers for MRD cells that may allow their identification for early detection of relapse;⑤At diagnosis, the proportions of N-Cadherin and Tie2 positive CD34+/CD38-/CD123+cells compartment were higher in cytogenetic unfavorable group, the white blood cell (WBC) count of more than 30×109/L and CD56 positive expression, which further proposed that N-Cadherin, Tie2 and CD44 expression may be associated with poor prognosis;⑥The proportions of N-Cadherin, Tie2 and CD44 positive CD34+/CD38-/CD123+ cells compartment were correlated with BM blast percentages at Post-Day 7, which proposed that N-Cadherin, Tie2 and CD44 co-expression may serve as markers for MRD cells that may allow their identification for early detection of relapse;⑦(87.75±6.01)%and (72.36±16.47)%AML1/ETO fusion signals were detected in the CD34+/CD38-/CD123+/N-cadherin+and CD34+/CD38-/CD123+/N-cadherin- LSCs populations, respectively. And (92.18±8.23)%and (80.87±16.45)%AML1/ETO fusion signals were detected in the CD34+/CD38-/CD123+/Tie2+ and CD34+/CD38-/CD123+/Tie2- LSCs populations, respectively, which suggesting that they might present at LSCs populations.⑧N-cadherin mRNA expression were detected in CD34+/CD38-/CD123+/N-Cadherin+ cells counterparts, and none in CD34+/CD38-/CD123+/N-Cadherin- cells counterparts, which would be as positive control. However, in 3 samples of Tie2 LSCs counterparts, N-cadherin mRNA expression was detected in both Tie2+ and Tie2- LSCs counterparts in 1 sample, but none in Tie2+ and Tie2- LSCs counterparts of another 2 samples. The results indicated that the expression of Tie2 and N-cadherin seems overlapped in some patients.⑨The relative quantity of MDR1 expression in N-cadherin+ and Tie2+ LSCs counterparts were higher than that of N-cadherin- and Tie2- LSCs counterparts(8.16±1.07 and 4.18±3.93 vs 0.06±0.06 and 0.46±0.49, respectively). These results could further confirm the finding that N-cadherin+ and Tie2+ LSCs counterparts are more resistant to chemotherapy than their negative LSCs counterparts.Conclusions:N-cadherin+ and Tie2+ expressed CD34+/CD38-/CD123+ LSCs populations could be less sensitive to chemotherapy and be enriched by chemoresistance. However, AML1/ETO and MDR1 were found in the N-cadherin+ and Tie2+ positive LSCs. It is suggested that N-Cadherin and Tie2 may be the potential markers of LSCs and also be the candidate therapeutic targets. Objective:G-rich oligonucleotides (GROs) have been demonstrated to inhibit proliferation and induce cell cycle arrest at S phase in tumor cell lines. The biological activity of GROs results from their binding to specific cellular proteins, nucleolin. Increases in nucleolin levels in unstressed cells led to activation of P53. It has been previously identified that activation of P53 could stimulate nucleolin-P53 complex formation under stress. GROs showed anti-proliferation activity through a non-antisense way, due to their stable G-quartets structure formation. This stable structure was found to interact with a specific cellular protein, nucleolin. Increases in nucleolin levels in unstressed cells led to activation of P53. And the biological activity of GROs results from their binding to nucleolin, as well as, nucleolin can interact with P53. The relationship among the effect of GRO, nucleolin and P53 is unknown yet. To investigate its mechanism of GRO, the effects of p53 on the biological function of GROs were studied.Methods:GROs and C-rich oligonucleotides (CROs) as control were performed to determine the function of GRO in leukemia cell lines. Western blot analysis was employed to identify the P53 protein expression in U937 cell lines, which have no P53 protein expression. HIV-based lentivector expression vector pCDH1-MCS1-EF1-copGFP containing full-length coding sequence (CDS) region of p53 was constructed and transfected to express p53 in U937 cell lines. Apoptosis assay by flow cytometry, cell cycle and MTT were performed to determine the function of GRO in U937 cell lines induced by the apoptotic stimuli, P53.Results:1) GRO was shown to have significant anti-proliferative activities, inducing apoptosis and cell cycle arrest at S phase in U937 cells (human monocytic leukemia cells) which have no P53 protein expression.2) By the laser scanning confocal microscope (LSCM) assay, we verify for the first time the co-localization between GRO and nucleolin inside U937 cells. However, P53 expression could cause the partial formation of nucleolin-P53 complexion by exposure to GRO.3) The effect of GRO was weakened in U937 cells with P53 expression.4) In U937 cells with GRO treatment, increased Cdk2 led the cells enter S phase. Then, Cdk2 began to decease that made the cells arrest in S phase. Expression of P53 in U937 cells affects the alteration of cell cycle and related regulation proteins. Conclusion:GRO was shown to have significant anti-proliferative activities, inducing apoptosis and cell cycle arrest at S phase in U937 cells. And in U937 cells with GRO treatment, increased Cdk2 led the cells enter S phase. Then, Cdk2 began to decease that resulted in the cells arrest in S phase. However, the effect of GRO was weakened in U937 cells with P53 expression, which may be related with the decreased intake of GRO because of the partial formation of nucleolin-P53 complexion.