Expression and clinical significance of antiapoptotic gene (survivin) in NB4 and acute promyelocytic leukemia cells.

To study survivin gene expression in APL cells and to explore its correlation with clinical manifestations. PML/RARα and survivin mRNA expression were analysed using RT-PCR. By treatment of ATRA, the survivin mRNA expression in NB4 cells gradually decreased with time and was almost undetectable in the 72th hour. Survivin was expressed in 67% of the 36 APL cases (de novo and relapse patients) with PML/RARα fusion gene expression. However, in 22 cases of remission stage patients without PML/RARα fusion gene expression, survivin was expressed in 36%. The survivin mRNA expression positive rate in de novo and relapse groups, and PML/RARα fusion gene L-type positive groups, was obviously higher than those in remission period groups and was significantly lower than those in acute leukemia groups. In 36 cases of de novo and relapse APL patients, all cases could obtain complete remission, irrespective of the survivin expression. APL patients expressed with survivin mRNA had DIC and serious infection (one patient died). The clinical symptom included slight skin or mucosa bleeding, fever, and asthenic for patients without the survivin mRNA expression. Later, two cases of APL patients with the survivin mRNA expression were treated by ATRA, induction differentiation sign in their peripheral blood and bone marrow figure was not obvious. It was concluded that the survive gene expression was lower in APL than those in any other types of leukemia, thus closely associated with clinical manifestation.

Homoharringtonine-induced apoptosis of human leukemia HL-60 cells is associated with down-regulation of telomerase.

Homoharringtonine (HHT), first isolated from the Chinese evergreen Cephalotaxus Harringtonia, has been shown inhibiting activity in leukemia in initial studies in China and in later studies in the US, but the detailed mechanism of action is still unclear. The goal of the experiments shown here is to explore the effect of HHT on the telomerase activity and apoptosis of human leukemia HL-60 cells. The telomerase activity of HL-60 cells was examined by the telomeric repeat amplification protocol (TRAP)--an enzyme-linked immunosorbent assay (ELISA). Apoptosis was analyzed by morphological observation, DNA agarose gel electrophoresis, flow cytometry (FCM), and TdT-mediated dUTP-biotin nick end labeling (TUNEL). After treatment with HHT at 5-500 microg/l for 48 hours, the level of telomerase activity in HL-60 cells decreased in a dose-and time-dependent manner. Simultaneously, HL-60 cells underwent apoptosis. In conclusion, our data suggest that HHT can inhibit the telomerase content of HL-60 cells effectively and induce apoptosis.

A study of the relationship between expression level of TRF1 protein and telomerase activity in human acute leukemia.

OBJECTIVE: To study the expression level of TRF1 (telomeric repeat binding factor 1) protein in human acute leukemia and relationship between expression level of TRF1 protein and telomerase. METHODS: A quantitative Western-Blot technique was developed using anti-TRF1(33-277) monoclonal antibody and GST-TRF1 purity protein as a standard to further determine the expression level of TRF1 protein in total proteins extracted from clinical specimens. RESULTS: Bone marrow tissues of 20 acute leukemia patients were studied, 11 healthy donors' bone marrows were taken as a control. The expression level of TRF1 protein was significantly higher (P<0.01) in normal bone marrow ((2.217+/-0.462) microg/microl) than that of acute leukemia patients ((0.754+/-0.343) microg/microl). But there was no remarkable difference between ALL and ANLL patients ((0.618+/-0.285) microg/microl vs (0.845+/-0.359) microg/microl, P>0.05). After chemotherapy, TRF1 expression level of patients with complete remission elevated ((0.772+/-0.307) microg/microl vs (1.683+/-0.344) microg/microl, P<0.01), but lower than that of normal ((2.217+/-0.462) microg/microl, P<0.01). There was no significantly difference after chemotherapy ((0.726+/-0.411) microg/microl vs (0.895+/-0.339) microg/microl, P>0.05). TRF1 expression level of patients with complete remission is higher than that of patients without complete remission ((1.683+/-0.344) microg/microl vs (0.895+/-0.339) microg/microl, P<0.01). All samples were determined for telomerase activity. It was confirmed that the activity of telomerase in normal bone marrow was lower than that of acute leukemia patients ((0.125+/-0.078) microg/microl vs (0.765+/-0.284) microg/microl, P<0.01). There was no significant difference of expression level of TRF1 protein between ALL and ANLL patients ((0.897+/-0.290) microg/microl vs (0.677+/-0.268) microg/microl, P>0.05). After chemotherapy, telomerase activity of patients with complete remission decreased ((0.393+/-0.125) microg/microl), but was still higher than that of normal ((0.125+/-0.078) microg/microl, P<0.01). CONCLUSION: The expression level of TRF1 protein has correlativity to the activity of telomerase (P<0.001).

[Impaired microfilament cytoskeleton rearrangement in cytomegalovirus infected cells].

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.

[Detection, enrichment and expansion of T lymphocytes mediating alloresponse based on cytokine].

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.

[Evaluation of interferon-gamma producing-cells using enzyme linked immunospot assay in mice model of acute graft versus host disease].

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.

[Study on unrelated donor allogeneic bone marrow transplantation with Bu-CY2 conditioning regimen for myelodysplastic syndrome].

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.

Induction of apoptosis by homoharringtonine in G1 phase human chronic myeloid leukemic cells.

BACKGROUND: Homoharringtonine (HHT) is a cephalotaxine ester derived from an evergreen tree found wildely throughout southern China, which has antileukemic activities against a variety of acute myeloid leukemic cells. For the sake of illustrating the mechanisms of HHT in the treatment of leukemia, we assessed the effect of HHT on the apoptosis of human chronic myeloid leukemic cell line K562. METHODS: The apoptosis of K562 cells induced by HHT was analyzed by transmission electron microscopy, agarose gel electrophoresis of DNA, flow cytometry and terminal deoxyribonucleotidyl transferase-mediated dUTP-biotin nick labeling. RESULTS: Characteristic apoptosis-related features emerged in K562 cells after exposed to HHT at a concentration 0.05-100 microg/ml. Transmission electron microscopy of HHT treated K562 cells displayed chromatin condensation and aggregation under the nuclear membrane, nuclear fragmentation and apoptosis body formation. Typical DNA ladder in agarose gel electrophoresis was observed in the cells exposed to HHT. The cell cycle analysis measured by flow cytometry showed G1 phase cells decreased with the increase of S phase cells while apoptosis was induced by HHT in K562 cells. The percentage of apoptotic cells in K562 cells treated with 50 microg/ml of HHT decreased significantly when pretreated with 1 microg/ml of cycloheximide, 0.05 microg/ml of Actinomycin D respectively. CONCLUSIONS: HHT has apoptotic effects on K562 cells. The HHT induced apoptosis mainly of the cells in G1 phase and this process required RNA transcription and protein synthesis.

Correlation between survivin mRNA expression and homoharringtonine induced apoptosis of malignant hematopoietic cells.

BACKGROUND: The inhibitor of apoptosis (IAP) gene family is involved in the suppression of apoptotic cell death as well as an increasing number of seemingly unrelated cellular functions. It is not known, however, whether IAP expression in malignant hematopoietic cells is affected by chemotherapeutic agents such as homoharringtonine (HHT). In this study, we investigated mRNA expression levels of IAPs, especially survivin, in various hematopoietic cell lines in relation with apoptosis induced by HHT. METHODS: Semiquantitative reverse transcriptase polymerase chain reaction was used to determine survivin mRNA levels. Cell apoptosis was examined by flow cytometry. Cell viability and proliferation assay was evaluated by MTT. The experiments were performed on the malignant hematopoietic cell lines MUTZ-1, K562, Jurkat, RMPI and HL60, with or without survivin antisense-oligodeoxynucleotides (AS-ODN) and HHT. RESULTS: The expression levels of survivin mRNA were variable in the cell lines and negatively correlated to HHT induced cell apoptosis. Survivin AS-ODN significantly decreased mRNA level of survivin, but not those of bax and bcl-2. Survivin also inhibited MUTZ-1 cell growth and induced apoptosis in a dose dependent manner. AS-ODN and HHT showed synergistic effect on MUTZ-1 cell growth. CONCLUSION: The apoptotic effect of HHT on the hematopoietic cell lines is associated with decreased level of survivin expression. Survivin could be a new marker for drug sensitivity and a new target for cancer treatment.

[Expression of C II TA gene in five human malignant hematological cell lines and its significance].

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.

Invasive fungal infection in allogeneic hematopoietic stem cell transplant recipients: single center experiences of 12 years.

Invasive fungal infection (IFI) is a growing cause of morbidity and mortality among patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We retrospectively reviewed the records of 408 patients undergoing allo-HSCTs during the period November 1998 to December 2009, analyzed the incidence and risk factors of IFI, and examined the impact of IFI on overall survival. A total of 92 (22.5%) episodes suffered proven or probable IFI (4 patients were proven, 88 patients were probable). Candida was the most common pathogen for early IFI, and mold was the most frequent causative organism for late IFI. A prior history of IFI, human leukocyte antigen (HLA) mismatch, long-time neutropenia, and acute graft-versus-host-disease (GVHD) were risk factors for early IFI. A prior history of IFI, corticosteroid therapy, cytomegalovirus (CMV) disease, and chronic GVHD were risk factors for late IFI. IFI-related mortality was 53.26%. The 12-year overall survival (OS) rate for IFI was significantly lower than that of patients without IFI (41.9% vs. 63.6%, P<0.01).

Human cytomegalovirus protects multiple myeloma cell line KM3 cells from apoptosis induced by growth factor withdrawal.

BACKGROUND: There is a higher rate of cytomegalovirus (CMV) reactivation in patients with multiple myeloma after an autologous stem cell transplantation, but no attention has been given thus far to a possible pathogenetic interplay between CMV and multiple myeloma. CMV can infect many kinds of cells, and CMV infection has been shown to inhibit apoptotic responses in several cell systems. In this study the authors investigated the alterations in apoptosis in the multiple myeloma cell line KM3 after infection with CMV and proposed a possible mechanism. METHODS: KM3 cells were infected with 100, 10, or 1 TCID50 of CMV and then cultured in serum-free RPMI 1640. An RT-PCR-based assay was used to detect mRNA expression of CMV-IE, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and IL-6 in CMV-infected and mock-infected cells. Flow cytometry was used to detect apoptotic cells. CMV particles and apoptotic cells were also examined with an electron microscope. RESULTS: CMV-infected KM3 cells clearly expressed immediate early (IE) antigen mRNA when compared to uninfected cells, and there were fewer apoptotic cells among cells treated with 100 or 10 TCID50 of CMV after culturing in serum-free RPMI 1640. CMV particles were observable in infected cells under an electron microscope. Expression of IL-6 mRNA increased after infection. CONCLUSION: CMV can infect the multiple myeloma cell line KM3, inhibit the apoptotic response in these cells after apoptosis induction in serum-free culture, and increase the expression of IL-6 mRNA.

[Detection and purification of cytokine secreting T lymphocytes in allogeneic reaction and a preliminary study on its clinical significance].

OBJECTIVE: Detection of cytokine secreting T lymphocytes after allogeneic peripheral blood mononuclear cell (PBMNC) stimulation was carried out and its clinical significance discussed so as to explore a new approach to study allogeneic reactive T lymphocytes. METHODS: Cytokine secretion assay (CKSA) was applied to detect T lymphocytes secreting interferon gamma (IFNgamma), interleukin-4 (IL-4) and IL-10 at single cell level in human mixed lymphocytes reaction. T cells secreting IFNgamma from PBMNC were detected in patients with acute graft versus host disease (aGVHD). RESULTS: In comparison with T cells secreting IL-4 and IL-10 which were (0.12 +/- 0.03)% and (0.10 +/- 0.03)%, respectively, a sizable proportion of T cells secreting IFNgamma could be detected (1.12 +/- 0.13)%. Preliminary result indicated that frequency of T cells secreting IFNgamma correlated with the occurrence of aGVHD. CONCLUSIONS: It is feasible to detect T lymphocytes secreting IFNgamma after allogeneic PBMNC and to apply CKSA technique for clinical research.

[The clinical significance of angiogenesis in the bone marrow of acute leukemia patients].

OBJECTIVE: To study the angiogenesis in bone marrow and the level of vascular endothelial growth factor (VEGF) in bone marrow fluid from acute leukemia (AL) patients and to explore their clinical significance. METHODS: Microvessels in each bone marrow specimen section were counted by immunohistochemical identification of microvascular endothelial cells with anti-human von Willebrand factor under light microscopy in field 400 times. The VEGF level in bone marrow fluid was measured by ELISA. RESULTS: Microvessel counts in untreated AL patients (22.82/x 400 field) were significantly higher than those in normal controls (7.17/x 400 field) and in the bone marrow remission (BMR) AL patients (8.57/x 400 field) (P < 0.001, both), while the controls in the latter were higher than those in controls (P < 0.05). There was no difference in microvessel counts between untreated AL patients and relapse/refractory AL patients (21.83/x 400 field) (P > 0.05). In the untreated AL group, the microvessel counts showed difference neither between 23 ALL patients (23.09/x 400 field) and 43 ANLL patients (22.37/x 400 field) (P > 0.05), nor between patients with M(1 - 3) (22.91/x 400 field) and M(4 - 5) (21.46/x 400 field) the two subtypes of FAB (P > 0.05). There was a positive correlation between the microvessel counts and the percentage of blast cells in the bone marrow (r = 0.311, P < 0.01). The VEGF level in the bone marrow fluid in untreated AL patients (188.88 ng/L) was significantly higher than that in BMR AL patients (78.74 ng/L) and controls (79.52 ng/L) (P < 0.01), while the latter two groups had no difference between each other (P > 0.05). The VEGF level in the bone marrow fluid from ANLL group (270.12 ng/L) was enhanced significantly compared with that from ALL group (101.70 ng/L) (P < 0.01) and associated with the microvessel counts (r = 0.464, P < 0.05). CONCLUSION: Microvessel counts increased in the bone marrow in patients with different types of AL, suggesting that angiogenesis might play an important role in the pathology of AL. Testing the VEGF level in the bone marrow fluid from ANLL patients could reflect the degree of angiogenesis and disease condition.

Homoharringtonine induces apoptosis of endothelium and down-regulates VEGF expression of K562 cells.

Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry. The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner. Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.

Human cytomegalovirus induces alteration of beta-actin mRNA and microfilaments in human embryo fibroblast cells.

OBJECTIVE: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on beta-actin mRNA and microfilaments. METHODS: HF cells shape was observed after the infection of CMV. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, beta-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. RESULTS: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of beta-actin mRNA decreased in a time- and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. CONCLUSION: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of beta-actin mRNA and the microfilaments.

TGF-beta1 treated murine dendritic cells are maturation resistant and down-regulate Toll-like receptor 4 expression.

OBJECTIVE: To explore the effects of transforming growth factor beta1 (TGF-beta1) on dendritic cells (DC). METHODS: Murine bone marrow cells were cultured with GM-CSF and TGF-beta1 to develop TGF-beta1-treated DC (TGFbeta-DC). Then they were stimulated by lipopolysaccharide (LPS). Their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using BrdU ELISA method and IL-12p70 protein was detected by ELISA. The expression of Toll-like receptor 4 (TLR4) was analyzed by semi quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and FCM. RESULTS: Compared to immature DC (imDC) cultured by GM-CSF alone, the TGFbeta-DC express lower CD80, CD86, I-Ab and CD40. The TGFbeta-DC were resistant to maturation with LPS. Maturation resistance was evident from a failure to up-regulate co-stimulatory molecules (CMs), to stimulate larger T cells proliferation and to enhance secretion of IL-12p70. We also found that TGF-beta1 could down-regulate TLR4 expression on TGFbeta-DC. CONCLUSION: TGFbeta-DC are resistant to maturation stimulus (LPS) and might have some correlation with the down-modulation of TLR4 expression.

[Inhibition effect of arsenic trioxide on the growth of human MDS cell line MUTZ-1 cells].

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.

[Expression of telomere repeat binding factor 1 (TRF1) protein in kidney cancer].

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.

[Detection and analysis of bcl-1/IgH rearrangement in mantle cell lymphoma]

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.