The effect of WT1 expression on the prognosis of allogeneic hematopoietic stem cell transplantation in acute leukemia / 中华血液学杂志

Objective: To study the effect of WT1 expression on the prognosis of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in acute leukemia (AL) and its significance as molecular marker to dynamically monitor minimal residual disease (MRD) . Methods: Retrospectively analyzed those AL patients who underwent allo-HSCT in the First Hospital Affiliated to Zhejiang University School of Medicine during Jan 2016 to Dec 2017, a total number of 314 cases, 163 males and 151 females, median age was 30 (9-64) years old. Comparing the difference of WT1 expression at diagnosed, pre-HSCT and after HSCT. Using the receiver operating characteristic (ROC) curve to determine the WT1 threshold at different time so as to predict relapse. The threshold of WT1 expression before transplantation was 1.010%, within 3 months after HSCT was 0.079% and 6 months after HSCT was 0.375%. According to these thresholds, WT1 positive patients were divided into low expression groups and high expression groups. Analyzed the relationship between overall survival (OS) , disease-free survival (DFS) , cumulative incidence of relapse (CIR) and WT1 expression. Results: The OS and DFS of high expression group pre-HSCT were lower than low expression group [69.2% (9/13) vs 89.1% (57/64) , χ(2)=4.086, P=0.043; 53.8% (7/13) vs 87.5% (56/64) , χ(2)=9.766, P=0.002], CIR was higher than low expression group [30.8% (4/13) vs 7.8% (5/64) , P=0.017]. There was no significant difference of OS and DFS between high expression and low expression group of 3 months after HSCT (P=0.558, P=0.269) . The OS and DFS of high expression group of 6 months after transplantation were both lower than low expression group (P=0.049, P=0.035) . Multivariate analysis showed that WT1>0.375% when 6 months after transplantation was the only independent prognostic factor for shorter DFS (P=0.022) . There was no statistically significant difference in CIR between the high-expression group and the low-expression group 3 months after transplantation and 6 months after transplantation (P=0.114, P=0.306) . Conclusion: High expression of WT1 before and after HSCT was an adverse prognosis factor. It is of clinical practical value to use WT1 as a transplant recommendation index for patients with acute leukemia and as a marker to monitor MRD dynamically.

Localization of human telomere repeat binding factor 1 in telomerase-positive and-negative cells and its expression during cell cycle / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To observe the distribution pattern of human telomere repeat binding factor 1(TRF1) in the telomerase-positive (HeLa) and telomerase-negative cells (WI38-2RA) and to investigate its expression level during the cell cycle.</p><p><b>METHODS</b>The full-length sequences of TRF1(TRF1FL) and its mutant with N and C terminus deletion (TRF1DeltaNC) were generated by PCR amplification, the resulting fragments were cloned into pEGFP-C2 mammalian expression vector. GFP-tagged proteins were verified by Western blotting with rabbit anti-TRF1 and mouse anti-GFP antibodies after cell transfection. Immunofluorescence staining were performed to detect the TRF1 localization in HeLa and WI38-2RA cells. Metaphase spreads from HeLa cells were also prepared to observe TRF1 localization in chromosomes. HeLa cells were arrested by thymidine and nocodazole at different cell stages. Cell cycles were analyzed by flow cytometry and TRF1 levels were evaluated by semi-quantitative Western blotting.</p><p><b>RESULTS</b>TRF1FL and TRF1PNC fragments were sized about 1.3 kb and 0.95 kb. GFP-tagged TRF1FL and TRF1DeltaNC proteins were 80 kD and 60 kD, respectively. In both HeLa and WI38-2RA cells, TRF1FL had a speckled distribution in the nuclei,however, TRF1FL did not coincide with promyelocytic leukemia (PML) nuclear body in HeLa cells while it exclusively did in WI38-2RA cells. Moreover, TRF1FL was exactly localized at the termini of metaphase spreads in HeLa cells. In contrast, TRF1PNC was diffusely distributed throughout the nuclei. Analysis by semi-quantitative Western blotting indicated that TRF1 levels increased with cell cycle progression, which reached the zenith at the M phase and went down to the nadir at G1/S point. The TRF1 level at M phase was about 3.9 times than that at G1/S point(t=12.92iP<0.01).</p><p><b>CONCLUSION</b>TRF1 has a different localization in telomerase-positive and telomerase-negative cells, which suggests TRF1 might exert different functions in these cells. TRF1 level is regulated with cell cycle.</p>

Telomerase activity of human bone marrow mesenchymal stem cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the telomerase activity in mesenchymal stem cells (hMSCs) from human bone marrow after their in vitro committed differentiation into adipocytes and cryopreservation.</p><p><b>METHODS</b>hMSCs were isolated from human bone marrow. The isolated hMSCs were induced to differentiate into adipocytes in vitro or cryopreserved. TRAP assay (telomerase repeat amplification protocol assay) was employed to detect telomerase activity in those hMSCs.</p><p><b>RESULTS</b>Telomerase activity (RTA) in hMSCs (n=19) was (1.46 +/-0.67)%, while that in hMSCs-derived adipocytes (n=3) was (11.80 +/-2.52)% (P<0.001). RTA of hMSCs-passage 1.3 (n=10) was (1.46+/-0.83)%, and that of hMSCs-passage 4-7(n=9) was (1.46 +/-0.47)% (P=0.99). Cryopreservation did not affect the telomerase activity in hMSCs, RTA of fresh hMSCs (n=13) was (1.41 +/-0.44)%, RTA of frozen hMSCs (n=6) was (1.57 +/-1.07)% (P=0.64).</p><p><b>CONCLUSION</b>hMSCs are telomerase-negative, but telomerase activity in hMSCs-derived adipocytes is upregulated.</p>

Expression of human telomere repeat binding factor 1 (TRF1) in acute leukemia cells and its correlation with telomerase activities / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the expression of human telomere repeat binding factor 1 (TRF1) to investigate the correlation of telomerase activity with acute leukemia.</p><p><b>METHODS</b>Leukemic cells were collected from 30 cases of acute leukemia. Realtime quantitative PCR with fluorescence probe hybridization was used to measure expression of TRF1 and hTERT mRNA in leukemic cells.</p><p><b>RESULTS</b>TRF1 mRNA expression was 0.0126 (0.0127-0.0546) in acute non-lymphocytic leukemia (ANLL), which was lower than that in normal mononuclear cells [0.0457 (0.00839-0.262), P<0.001], but its expression in acute lymphoblastic leukemia (ALL) cells [0.0745 (1.92 x 10(-6)-0.193)] had no significant difference compared with that in normal mononuclear cells. TRF1 expression in ANLL cells was significantly lower than that in ALL cells (P=0.001). The expressions of TRF1 mRNA in AL cells and normal mononuclear cells had no significant correlation with expression of hTERT mRNA (r=-0.173, P=0.207).</p><p><b>CONCLUSION</b>The expression of TRF1 is lower in ANLL cells, which indicates TRF1 may have some effect on telomerase activity by regulating telomere length in ANLL cells.</p>

Expression of Pin1 in malignant hematopoietic cells and its relation with cell cycle / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the expression of peptidyl-prolyl cis/trans isomerase (PPIase or Pin1) in malignant hematopoietic cells and its relation with cell cycle.</p><p><b>METHODS</b>Realtime quantitative PCR with fluorescence probe hybridization was used to measure expression of Pin1 mRNA in malignant hematopoietic cell lines and normal mononuclear cells separated from bone marrow. HeLa cells were blocked with Thymidine and Nocodazole in different cell phases and then the expression of Pin1 mRNA and protein were detected by realtime-PCR and immunoblotting.</p><p><b>RESULTS</b>The expression of Pin1 in malignant hematopoietic cell lines was significantly higher than that in normal controls (0.339 +/-0.093 compared with 0.038 +/-0.005, P<0.01). Its expression in myeloid malignant hematopoietic cell lines was significantly higher than that in normal controls (0.388 +/-0.115 compared with 0.038 +/-0.005, P<0.01) and so was the malignant lymphocytic cell lines (0.226 +/-0.166 compared with 0.038 +/-0.005, P<0.01). The expression of Pin1 was closely correlated with cell cycle. It was the highest in G1 phase and the lowest in S phase (110.762 +/-16.737 compared with 4.080 +/-0.634, P<0.01).</p><p><b>CONCLUSION</b>Pin1 is overexpressed in malignant hematopoietic cell lines and its expression is different during cell cycle that is highest in G1 phase and lowest in S phase.</p>