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Objective: To investigate the effect of cytomegalovirus (CMV) infection on actin and microfilament in human embryo fibroblast cells (HF) and its relationship with CMV replication., Methods: Cell morphology was observed after the infection of CMV. Western-blot was used to measure the expression levels of beta-actin, G-actin and F-actin proteins. CMV immediately early antigen (CMV IE) in HF cells was analyzed by indirect immunofluorescence assay. Microfilament alteration was determined by cytoskeleton fluorescence probe., Result: CMV IE was demonstrated in more than 95% of HF cells after infection, which was primarily located in nucleus. The shape of HF cells changed from thin shuttle like to round and thick ball like, even escaping from wall after infection by CMV. Compared with control group, the expression of G-actin protein increased at 24 h of CMV infection (0.941 +/-0.061 compared with 0.714 +/-0.119, P <0.05), then decreased at 72 h, 96 h respectively(0.218 +/-.035, 0.230 +/-0.055 compared with 0.714 +/-0.119, P <0.05). The levels of F-actin in infected cells gradually decreased at 24 h, 72 h and 96 h compared with control HF cells (0.256 +/-0.021, 0.127 +/-0.032, 0.026 +/-0.008 compared with 0.373 +/-0.050, P<0.05). In infected HF cells, microfilaments were found ruptured, arranged turbulently. Cells fused and fluorescence density of microfilament markedly reduced., Conclusion: Cytomegalovirus can induce alteration of actins and microfilament, which may be associated with its infection, replication and reactivity in host cells.

Objective: To investigate IFN-gamma producing-cells (IFN-gamma PCs) in allogeneic mixed lymphocyte reaction (MLR) and acute graft versus host disease (aGVHD) model of mice., Methods: Enzyme linked immunospot assay (ELISPOT) was applied to study IFN-gamma PCs in MHC mismatched mice spleen cell MLR and aGVHD model of mice., Result: IFN-gamma PCs increased significantly in MLR after allogeneic mice spleen cell stimulation. In the experimental mice aGVHD model, IFN-gamma PCs were significantly higher in the severe aGVHD group than those in the moderate aGVHD. In the moderate aGVHD group, mice with GVHD prophylaxis regimen demonstrated significantly lower level of IFN-gamma PCs, compared with those without prophylaxis. IFN-gamma PCs were significantly correlated with the GVHD clinical scores in the group with moderate aGVHD and prophylaxis regimen., Conclusion: ELISPOT is a fast, sensitive and specific approach to evaluate alloresponse in allogeneic mice MLR and IFN-gamma PCs are correlated closely with the severity of aGVHD and prophylaxis regimen in the MHC-mismatched mice model.

Objective: To evaluate the efficacy and safety of Bu-CY(2) conditioning regimen on allogeneic bone marrow transplantation (BMT) with unrelated donor for myelodysplastic syndrome., Methods: Six patients received chemotherapy regimen of busulfan (Bu) and cyclophosphamide (CY) before allogeneic BMT (Bu 4 mg . kg(-1) . d(-1), -7 d - -4 d, CY 60 mg . kg(-1) . d(-1), -3 d - -2 d). Mycophenolate mofetil combined with cyclosporin A and methotrexate was used for prevention of acute graft-versus-host disease after transplantation. Lipo prostaglandin E(1)was used in prophylactic regimen for hepatic veno-occlusive disease., Result: Neutrophil count began to be higher than 0.5 x 10(9)/Lat the 18th day after BMT. Platelet count began to be higher than 20 x 10(9)/Lat the 21st day after BMT. Disease-free survival in the six patients was 27 months., Conclusion: Bu-CY(2) conditioning regimen on allogeneic bone marrow transplantation with unrelated donor is an effective therapy for patients with myelodysplastic syndrome.

Objective: To detect, enrich and expand the cytokine secreting T lymphocytes after allogeneic PBMNCs stimulation., Methods: The novel cytokine secretion assay (CKSA) was applied to detect T lymphocytes secreting IFN-gamma at single cell level in human mixed lymphocytes reaction. IFN-gamma secreting T cells were enriched by means of magnetic sorting system and expanded with OKT(3), anti-CD(3)mAb and IL-2 combination. Antigen specificity of the expanded cells was confirmed using enzyme linked immunospot assay., Results: A sizable proportion of IFN-gamma secreting T lymphocytes could be detected [(1.12 +/-0.13)% compared with (0.23 +/-0.07)%] and be further enriched to (67.3 +/-10.5)%, or (93.8 +/-22.1) fold. T lymphocytes could be expanded up to 600-fold within 21-28 days and the specific IFN-gamma response of expanded cells was confirmed with stimulation of the relevant allogeneic PBMNC, which was significantly higher than the irrelevant PBMNC control., Conclusion: It is feasible to detect significantly increased IFN-gamma secreting T lymphocytes after allogeneic PBMNCs stimulation based on the CKSA technique at single cell level and these cells can be efficiently enriched and expanded for further research.

Objective: To investigate the role of MHC class II Transactivator (C II TA) in expression of HLA molecules in five human malignant hematological cell lines., Methods: The expressions of HLA molecules and C II TA protein were detected by immunohistochemistry and flow cytometry. The expression of C II TA gene was detected by RT-PCR. The response of peripheral T cells after stimulation by Jurkat cells was detected by mixed lymphocyte reaction., Result: The HLA II-positive tumor cells expressed the C II TA and IFN-gamma induced the expression of HLA I, II in tumor cells, which were able to express C II TA constitutively., Conclusion: There is a correlation between the inability of the tumor cells in response to IFN-gamma for HLA expression and the deficiency in the inducible expression of C II TA.

Objective: 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., Methods: 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., Results: 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)., Conclusion: 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.

Objective: To isolate and identify TRF1 immunoprecipitating protein complex and to clone the candidate gene., Methods: The co-immunoprecipitation assay was employed to isolate TRF1 protein complex and the immunoprecipitate was subjected to MALDI-TOF mass spectrometry for protein identification. The candidate gene was amplified by temperature-gradient PCR from human testis cDNA library and then cloned into pEGFP-C2 vector for eukaryotic expression. The amplified gene was verified by direct sequencing and GFP-tagged protein was confirmed by immunoblotting., Results: Tara protein with the size of 68 kD was identified from the TRF1 precipitate. The candidate gene amplified from cDNA library was about 1.7 kb as expected. Sequencing demonstrated the amplified fragment had 99.9% of homogenesis with Tara CDS sequence (gi:30474869). GFP-tagged fusion protein was about 100 kD. Tara was diffusely distributed in cytoplasm at interphase and in whole cells at mitotic phase., Conclusion: Tara might be an interacting protein with TRF1. However, further investigation would be required to confirm if they were bona fide partners.

Objective: To study the expression of peptidyl-prolyl cis/trans isomerase (PPIase or Pin1) in malignant hematopoietic cells and its relation with cell cycle., Methods: 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., Results: 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)., Conclusion: 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.

Objective: To investigate the telomerase activity in mesenchymal stem cells (hMSCs) from human bone marrow after their in vitro committed differentiation into adipocytes and cryopreservation., Methods: 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., Results: 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)., Conclusion: hMSCs are telomerase-negative, but telomerase activity in hMSCs-derived adipocytes is upregulated.

Objective: To investigate the expression levels of telomere repeat binding factor 1(TRF1) protein in normal kidney tissue and kidney cancer., Methods: Specimens of kidney cancer and pericancerous tissues were collected from 32 cases of renal carcinoma. A quantitative Western blotting technique was developed using TRF1 monoclonal antibody to determine the expression level of TRF1 protein in total protein extracts from tissue specimens., Results: The expression level of TRF1 protein was higher in normal kidney tissues (3.611 +/-1.922 microg/microl) than that of cancer tissues (2.428 +/-1.352 microg/microl) (t=5.776, P<0.01)., Conclusion: The expression level of TRF1 protein is significantly reduced in kidney cancer and the level is negatively correlated with malignant degree of the cancer.

Objective: To study the expression of human telomere repeat binding factor 1 (TRF1) to investigate the correlation of telomerase activity with acute leukemia., Methods: 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., Results: 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)., Conclusion: 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.

Objective: To provide experimental basis for extending the indications of arsenic trioxide (As(2)O(3)) in clinical application., Methods: MTT assay was used to detect the cytotoxicity of As(2)O(3) in combination with daunorubicin (DNR), cytosine arabinoside (Ara-C), harringtonine (H) and vincristine (VCR) respectively on leukemic cells from 23 newly diagnosed cases with acute non-promyelocytic leukemia (ANPL) and 16 cases of relapsed, refractory ANPL., Results: (1) As(2)O(3) inhibited the growth of leukemic cells from both newly diagnosed or relapsed and refractory ANPL patients, and there was no statistical difference in cytotoxicity of the patients in the two groups [(12.6 +/-7.7 compared with 10.1 +/-6.2)%, P<0.05]. (2) There was no correlation between the cytotoxicities of As(2)O(3) and Ara-C, H or VCR (P<0.05), but a linear correlation between As(2)O(3)and DNR was found (r=0.432, P<0.05).(3) Additivity and synergism of the cytotoxicity was found in most of the ANPL patients when As(2)O(3) was combined with the four chemotherapy drugs and the combination of As(2)O(3) with DNR or VCR enhanced the cytotoxicity significantly (P<0.05)., Conclusion: The results indicate that As(2)O(3) might be used in treatment of newly diagnosed or relapsed and refractory ANPL patients;and the combination of As(2)O(3) with DNR or VCR may enhance its therapeutic efficacy.

Objective: To investigate the inhibition effect of arsenic trioxide (AS(2)O(3)) on the growth of human MDS-RAEB cell line MUTZ-1 cells and to explore the possible cellular and molecular mechanisms., Methods: The apoptosis and differentiation of MUTZ-1 cells induced by AS(2)O(3) solution of different concentrations were studied with cell morphology, MTT, DNA fragmentation assay, RT-PCR, Nitroblue tetrazolium (NBT) reduction method and flow cytometry., Result: (1) Low concentration ofAS(2)O(3) (0.05 - 0.25 micromol/L) had no marked growth inhibition effect on MUTZ-1 cells; after 14 d treatment, it down-regulated the expression of positive cell differentiation antigens CD38, CD7, CD10, HLA-DR (P<0.05), but did not up-regulate the expression of CD11b (P>0.05). (2) After treatment with 1.0 - 20.0 micromol/L of AS(2)O(3), MUTZ-1 cells presented typical features of apoptosis with a dose dependent manner (r=-0.999, P<0.05). The expression of bcl-2 mRNA and the ration of bcl-2/bax were decreased after AS(2)O(3) treatment (P<0.05)., Conclusion: Low concentration of (2)O(3) may have partial differentiation inducement on MUTZ-1 cells. With a certain range of dose (1.0 - 20.0 micromol/L), (2)O(3) can induce apoptosis of MUTZ-1 cells. (2)O(3) can significantly down-regulate bcl-2 and it might be one of the mechanisms of (2)O(3) treatment.

OBJECTIVE: To study the mechanism of inhibitory effect of Ubenimex on human leukemic cells. METHODS: K562 and HL60 cells were treated with Ubenimex at different concentrations, and the growth inhibition was analysed by MTT assay. Cell apoptosis was evaluated by light microscopy, agrose gel electrophoresis, TUNEL labeling method and flow cytometry (FCM) assay. RESULTS: (1)Treatment with Ubenimex remarkably inhibited the growth of HL60 cells, the IC(50) of Ubenimex for HL60 cells was 13.03&mgr;g/ml. But K562 cells were less sensitive than HL60. Ubenimex inhibited the growth of HL60 and K562 cells in a dose-dependent manner. (2)Apoptosis of leukemic cells was induced by Ubenimex, which was shown by the changes in morphology, DNA ladder on agrose gel, TUNEL labeling,typical peak before G1 phase of cell cycle and the positive of Annexin V(FITC) on the cells membrane with FCM. (3)Ubenimex induced apoptosis of K562 and HL60 cells in a dose-and-time-dependent manner. (4)The cell cycle analysis by FCM showed that the HL60 cells were blocked in G1 phase after treated by Ubenimex. Conclution Ubenimex can efficiently induce apoptosis of HL60 and K562 cells, this may be one of the mechanisms for inhibiting effect of Ubenimex on leukemia.

OBJECTIVE: To investigate the specificity and sensitivity of the PCR technique in the identification of bcl-1/IgH gene rearrangement in mantle cell lymphoma (MCL) and to characterize the bcl-1/IgH junction DNA sequences. METHODS: A semi-nested PCR method to amplify bcl-1/IgH gene rearrangement in DNA from fresh frozen lymphonode specimen was established. Twenty-eight cases of mantle cell lymphoma were analyzed for the presence of bcl-1/IgH gene rearrangement. The rearrangement products was cloned and sequenced to recognize the junction sequences, the breakpoints in the bcl-1 region, and J(H) gene involved in the rearrangement. RESULTS: A bcl-1/IgH gene rearrangement was detected in 17 out of 28 cases of MCL, while only 9 cases was detected with single step PCR method (X(2)=4.59, P<0.05). The rearrangement product varied in size between 74 to 162 base pairs, and the length of the junction sequences ranged from 6 to 24 base pairs. Ten different bcl-1 breakpoints were clustered within 65 base-pair spans, among which, 5 breakpoints (located at 430, 440, 451, 486 and 492) were never reported by other authors. The most common J(H) gene segments utilized in the translocation were J(H) 4 (8/18), then J(H)5 (3/18), J(H)6 (2/18), J(H)4/5 (2/18). J(H)1 2/18, and J(H)3 (1/18). CONCLUSION: These results indicate that the semi-nested PCR is a specific and sensitive method for the detection of bcl-1/IgH gene rearrangement in mantle cell lymphoma, which has implications for both the diagnosis and clinical management of mantle cell lymphoma. The recognization the potential mechanism of bcl-1/IgH gene rearrangement will help us to know the exact pathogenic machanisms of MCL.