[Effects of specific siRNA targeting at bcr-abl fusion gene and its combination with p27 gene clone on chronic myeloid leukemia cell line K562].

This study was purposed to explore the effect of specific small interfering RNA targeting at bcr-abl fusion gene and its combination with p27 gene clone on the proliferation, cell cycle and apoptosis of chronic myeloid leukemia (CML) cell line K562. CML cell line K562 was used as the study object. A 21nt siRNA targeting at the fusion site of b3:a2 mRNA in bcr-abl fusion gene was designed, synthesized and transfected into the K562 cells as RNA interference group. Northern blot was used to detect the bcr-abl fusion gene, Western blot was used to detect the expression of P210 protein and apoptosis-related protein BCL-xL after the transfection. Meanwhile, p27 gene was amplified from peripheral blood mononuclear cells by RT-PCR, and was confirmed to be correct by sequencing, then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line by Lipofectin. After being selected with G418, p27-pcDNA3.1-K562 cell clone stably expressing p27 was isolated. P27 protein was identified by Western blot. Finally, siRNA and p27 gene clone were together applied to K562 cells, the cell survival rate was tested by MTT. The cell cycle and the apoptosis were tested by flow cytometry. The result showed that in contrast with the control group, the expression level of bcr-abl fusion gene was much lower in siRNA group, about 18.4% of K562 cells in siRNA group were apoptotic at 24 hours after siRNA transfection, and the expression of apoptosis-associated protein BCL-xL was greatly down-regulated. The expression of P27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. The strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells, as compared with control K562 cells. The count of p27-pcDNA3.1-K562 cells in G(0)/G(1) phase increased apparently, but that in S phase declined greatly. Cell cycle was arrested in G(0)/G(1) phase. After the combination of p27-pcDNA3.1-K562 cells with specific siRNA, the percentage of apoptosis obviously increased and cell survival rate significantly declined. It is concluded that the specific siRNA distinctly inhibits the expression of bcr-abl fusion gene, and can induce K562 cell apoptosis. The combination of specific siRNA with P27 gene clone displays a synergy of inhibition and pro-apoptosis effects to K562 cells.

[Role of mitochondria pathway in signal transduction of chronic myeloid leukemia].

This study was aimed to investigate the role of mitochondria pathway in signal transduction of chronic myeloid leukemia (CML). After bcr3/abl2 antisense oligodeoxynucleotide (ASO) was introduced into CML cell line K562 cells by liposomal transfection, the cell viability was detected by MTT assay, the cell apoptosis was determined by flow cytometry (FCM), the mitochondrial membrane potential (DeltaPsi) was labeled by Rhodamine 123 and examined by FCM, and the expression of mitochondrial apoptosis signal transduction pathway related proteins cytochrome C was analyzed by Western blot. The results showed that after K562 cells were exposed to 2 micromol/L of bcr3/abl2 ASO for 24 hours, bcr3/abl2 ASO significantly inhibited cell viability with inhibitory rate of 65.7%, induced the apoptosis of K562 cell line with apoptotic rate of 16.9%, and decreased mitochondrial Deltapsi of K562 cells with the reducing rate of 38.33%, enhanced the expression of cytochrome C with increase of optical density value from 2.33 +/- 0.3 to 4.78 +/- 0.1 by laser photometric scanning. It is concluded that mitochondria pathway plays an important role in signal transduction of chronic myeloid leukemia by directing apoptotic signal transduction.

[Effect of tyrosine-kinase Inhibitor on p15 gene transfected K562 cells].

The objective of study was to investigate the combined effect of tyrosine-kinase inhibitor (imatinib) and p15 gene on the proliferation, cell cycle and apoptosis of chronic myeloid leukemia cell line K562. p15 gene was amplified from peripheral blood mononuclear cells by RT-PCR, and confirmed by DNA sequencing, then the recombinant p15-pcDNA3.1 vector was constructed and transfected into K562 cell line by Lipofectine. After screening with G418, p15-pcDNA3.1-K562 cell clone stably expressing P15 was isolated. P15 protein was identified by Western blot. The cell survival rate was determined by MTT, cell cycle and apoptosis were detected by flow cytometry. The results showed that partial deletion of p15 gene in K562 cells was verified by DNA sequencing, leading to the function of P15 protein to be lost. The expression of P15 protein can be detected by Western blot in p15-pcDNa3.1-K562 cells. A strong inhibition of cell proliferation was observed in p15-pcDNA3.1-K562 cells as compared with that of the control K562 cell. The cells of G(0)/G(1) phase in p15-pcDNA3.1-K562 cells increased apparently, and S phase cells declined signifcantly. Cell cycle was arrested in G(0)/G(1) phase. The percentage of apoptotic cells greatly increased after transfection with p15-pcDNA3.1-K562 cells combined with imatinib, and cell survival rate notably declined. It is concluded that the imatinib in combination with the expression of p15 gene has a synergistic effect on the inhibition of K562 cell proliferation and promotion of its apoptosis.

Immune protection effect of exosomes against attack of L1210 tumor cells.

We investigated the anti-tumor immunity of L1210 cell-secreted exosomes. Exosomes were purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. Expression of H-2D and interstitial cell adhesion molecule (ICAM(-1 was investigated by solid-phase immuno-electron microscopy and expression of Hsp70 was investigated by western blotting. DBA/2 mice were immunized with a given dose of exosomes (2.5 or 5 microg(. Transmission electron microscopy revealed that L1210-derived exosomes were membrane vesicles. They were labeled by colloidal gold H-2D and ICAM-1. Western blot analysis demonstrated the presence of Hsp70 antigens in L1210 exosomes. Exosome immunization partly inhibited the growth of implanted tumor in mice. There was a significant difference between exosome groups and the control group (P < 0.05(. In conclusion, exosomes can be considered as a candidate therapeutic vaccine for leukemia.

[The effects of Th1/Th2 cells on in vitro expansion and colony forming of CD34+ cells of aplastic anemic patient].

AIM: To investigate the effects of both Th1/Th2 imbalance and its re-attainment on in vitro expansion and hematopoiesis of CD34+ cells from a severe aplastic anemia (sAA) patient. METHODS: A preliminary-diagnosed sAA patient was studied. (1) Bone marrow mononuclear cells (BMMNC) were isolated and CD34+ and CD4+ cells were enriched by magnetic beads respectively. (2) The Th1/Th2 cell ratio within CD4+ subset was detected by flow cytometry (FCM). (3) Enriched-CD34+ cells were expanded and re-enriched for enough cells to be divided into four groups: control, Th cell treatment, Th cell and IFN-gamma treatment, and Th cell and IL-4 treatment, respectively. (4) After expansion for 10 d, colony-forming unit assay was performed on cells in each group. (5) Patient's Th1/Th2 ratio was followed up by FCM after immunotherapy. RESULTS: (1) Symptom remission was achieved after therapy for 5 months. (2) Th1/Th2 cell ratio of the patient before and after remission was 22.47 and 12.27, respectively, while that of healthy controls was 8.98+/-4.45. (3) The CD34+ cell expansion rates as well as CFU numbers, from high to low, were ranked as control, Th cell and IL-4 contained group, Th cell contained, and Th cell and IFN-gamma contained group. CONCLUSION: Predominant Th1 cells seem to directly inhibit the self-renewal, proliferation and lineage differentiation of CD34+ cells in vitro, which can be counteracted by IL-4, probably mediated by switching Th1/Th2 balance.

[The diagnostic significance of Ig heavy chain rearrangement detected in B cell lymphoma patients' serum or plasma blood samples].

OBJECTIVE: To evaluate the diagnostic significance of detecting immunoglobulin (Ig) heavy chain (IgH) by using serum or plasma as blood samples. METHODS: First, collect serum and plasma blood samples of patients with B-NHL and extract tumor-derived DNAs. Then design the primer to amplify framework3 (Fr3) from the V segment regions to the J regions of the IgH complementary determining region III (CDR-III) gene. Detect the positive ratio of IgH rearrangement with PCR and evaluate its diagnostic significance. RESULTS: B lymphoma cell line Raji was used as the positive control. Of the 30 B-NHL cases diagnosed with morphologic study, 25 (83.3%) showed IgH rearrangement. DNAs extracted from healthy adults and chronic lymphadenitis patients showed negative result. There was no statistical pertinence between IgH gene rearrangement and clinical manifestation, clinical staging and tumor burden. CONCLUSION: Tumor-derived DNA can be detected in serum or plasma of the majority of patients with B-cell lymphoma. Testing of serum or plasma for tumor associated DNA may be a novel parameter for clinical diagnosis without the restriction of the position of enlarged lymph node.

Effects of STI571 and p27 gene clone on proliferation and apoptosis of K562 cells.

AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line. METHODS: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line. p27-pcDNA3.1-K562 cell clone was screened by G418 after transfection, p27 protein was identified by Western blot. MTT was used to detect the survival rate of the cell. Flow cytometry was used to detect cell cycle and apoptosis index. RESULTS: The expression of p27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. A strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells as compared with that of the control (pcDNA3.1-K562 cells). The cells at G0/G1 phase were significantly increased, and cells at S phase were greatly declined. The apoptosis index was increased greatly after p27-pcDNA3.1-K562 cells were treated with STI571, and survival rate of the cell was markedly declined (0.35-0.58, P<0.05-0.048 vs STI571-K562 cell, 0.35-0.72, P<0.01-0.001 vs p27-K562 cell). CONCLUSION: p27 and STI571 have a synergistic action on inhibition of proliferation and induction of apoptosis on K562 cells.

[Effect of specific siRNA targeting against bcr-abl chimeric gene on chronic myelogenous leukemia cells].

OBJECTIVE: To investigate the effect of specific small interfering RNA (siRNA) targeting against bcr-abl chimeric gene on the biological traits of chronic myelogenous leukemia (CML) cells. METHODS: CML cells of the line K561 transcribing a type of b3: a2 mRNA of bcr-abl chimeric gene were cultured. A 21nt siRNA targeting against the chimeric location of the b3: a2 mRNA of bcr-abl chimeric gene was designed, synthesized, and transfected into the K562 cells as RNA interference group. Another K562 cells were transfected with fluorescein enzyme gene specific siRNA as indifferent controls, or with lipid alone as blank vector controls. Some K562 cells without treatment were used as normal controls. 48 hours after the transfection Western blotting was used to detect the expression of P210bcr-abl fusion protein. 3H-TdR incorporation was used to detect the proliferation activity of K562. Annexin V-fluorescencein isothiocyanate (FITC)/phosphatidylinositol (PI) staining was used to detect the apoptosis of K2562 cells. Flow cytometry was used to observe the cell cycle of K562 cells. Benzidine staining was used to detect the differentiation of K562 cells towards erythrocytic series. Western blotting was used further to detect the expression of apoptosis-related protein Bcl-xL/Bax. RESULTS: (1) In contrast with the control groups, the expression level of bcr-abl chimeric gene was much lower in the RNAi group. (2) (3)H-TdR incorporation test showed time-dependent inhibition of proliferation of K562 cells, reflected in decrease of counts per minute (CPM) value in RNAi group 24 h, 48 h, 72 h, and 96 h after siRNA transfection by 33.06%, 52.25%, 57.64%, and 70.87% respectively (F=17.7, P < 0.01). (3) About 43.2% of K562 cells in the RNAi group were apoptotic 48 h after siRNA transfection (F=13.6, P < 0.01). (4) In contrast with the control groups, the expression of apoptosis-associated protein Bcl-xL was greatly down-regulated; however, the expression of Bax protein showed little change. (5) The percentage of benzidine-positive cells in the RNAi group was 23.5% +/- 3.2%, significantly higher than those in the indifferent control group, blank vector group, and normal control group (2.4% +/- 0.3%, 4.5% +/- 0.5%, and 3.6% +/- 0.2% respectively, all P < 0.01), which meant that part of the K562 cells differentiated towards erythrocytic series. (6) The percentage of G1 phase of K561 cells in the RNA1 group was significantly higher than those of the other groups (F = 6.2, P < 0.05), showing a capture in G1-phase of cell cycle. CONCLUSION: The specific siRNA distinctly inhibits the expression of bcr-abl chimeric gene and influences essential biological traits of K562 cells, which will ultimately result in differentiation or apoptosis of K562 cells.

Expression of COX-2, iNOS, p53 and Ki-67 in gastric mucosa-associated lymphoid tissue lymphoma.

AIM: To assess the expression of cyclooxygenase-2 (COX-2), nitric oxide synthase (iNOS), p53 and Ki-67 in gastric mucosa-associated lymphoid tissue (MALT) lymphoma and clarify the relationship between COX-2 expression and iNOS or p53 expression in these patients. METHODS: The expressions of COX-2, iNOS, p53 and Ki-67 were detected in 32 gastric MALT lymphoma specimens and 10 adjacent mucosal specimens by immunohistochemical Envision method. RESULTS: COX-2 and iNOS expressions were significantly higher in gastric MALT lymphoma tissues than those in adjacent normal tissues. The expression of COX-2 was observed in 22 of 32 cases of MALT lymphoma tissues (68.8%). A positive cytoplasmic immunoreactivity for iNOS was detected in 17 of 31 cases (53.1%). COX-2 expression in gastric MALT lymphoma tissues was positively correlated with iNOS expression (r=0.448, P=0.010) and cell proliferative activity analyzed by Ki-67 labeling index (r=0.410, P=0.020). The expression of COX-2 protein did not correlate with age, sex, stage of disease, lymph node metastasis or differentiation. The accumulation of p53 nuclear phosphoprotein was detected in 19(59.4%) of tumors. p53 protein was expressed in 11 of 23 assessed LG tumors and in 8 of 9 assessed HG tumors. The difference of p53 positivity was found statistically significant between LG and HG cases (P=0.0302). The p53 accumulation correlated with advanced clinical stage (stage III+IV vs stage I+II, P=0.017). There was a significant positive correlation between COX-2 expression and p53 accumulation status (r=0.403, P=0.022). The mean PI of Ki-67 in each grade group were 36.0+/-7.73% in HG and 27.4+/-9.21% in LG. High-proliferation rate correlated with HG tumors (r=0.419, P=0.017). The correlation coefficient showed a significant positive correlation between PI and COX-2 expression in MALT lymphoma patients (r=0.410, P=0.020). CONCLUSION: COX-2 expresses in the majority of gastric MALT lymphoma tissues and correlates with cellular proliferation and iNOS expression. COX-2 overexpression is closely associated with p53 accumulation status. iNOS and COX-2 may play a synergistic role in the pathogenesis of gastric MALT lymphoma.

[Survivin antisense oligonucleotide induces lymphoma cells apoptosis and sensitizes the cells to chemotherapy].

OBJECTIVE: To explore the effect of antisense oligodeoxynucleotide (ASODN) of survivin gene on apoptosis and chemotherapy sensitivity of lymphoma cell line Raji. METHODS: Anti-survivin phosphorothioate ASODN was synthesized and transfected into Raji cells by lipofectin. MTT assay was used to detect cytotoxicity. Apoptosis was observed by fluorescence microscopy and flow cytometry. Survivin expression was determined by RT-PCR and Western-blotting. RESULTS: (1) survivin ASODN inhibited the cells proliferation in a dose and time dependent manner. (2) A higher apoptosis rate (33.0%) could be induced in Raji cells by survivin ASODN as compared with that induced by the sense oligodeoxynucleotide (11.5%) (P < 0.05). (3) The expression of survivin mRNA and protein significantly decreased after treatment with survivin ASODN. (4) There was a significant increase of cell inhibition rate after exposure to the combination of survivin ASODN and Vm26 as compared to Vm26 or survivin ASODN alone (both P < 0.05). CONCLUSION: Survivin ASODN is able to inhibit the proliferation of Raji cells, induce the apoptosis, and enhance the sensitivity of Raji cell to chemotherapy via specific down-regulation of survivin expression.

[Expression of survivin and caspase-3 in non-Hodgkin's lymphoma and their clinical significance].

BACKGROUND & OBJECTIVE: Survivin, a member of the inhibitor of apoptosis protein (IAP) family, can directly inhibit caspase-3 and caspase-7 activity and plays an important role in oncogenesis. This study was designed to investigate the expression of survivin and caspases-3 in non-Hodgkin's lymphoma (NHL) with different aggressiveness and their clinical significance. METHODS: The expression of survivin and caspase-3 in 54 cases of NHL were determined with immunohistochemistry of EnVision. RESULTS: The expression rates of survivin and caspase-3 were 51.9% (28/54) and 83.3% (45/54), respectively. The expression of survivin in NHL patients with low grade malignancy (19%, 4/21) was lower than that of NHL patients with intermediate-high grade malignancy; the difference was statistically significant. The expression of caspase-3 showed the same tendency. Co-expression rate of survivin and caspase-3 was 46.3% (25/54). CONCLUSION: The expression of survivin is upregulated in NHL.

[Experimental research on tyrosine-kinase inhibitor STI571 and P21WAF gene clone to treat chronic myeloid leukemia].

To explore the effect of a tyrosine-kinase inhibitor STI571 and P21(WAF) gene clone on the proliferation, cycle, apoptosis of leukemia cell line K562, P21(WAF) gene was obtained by RT-PCR, and its sequence was approved to be correct, then P21-pcDNA3.1 vector was constructed and transfected into K562 cell line. After selected with G418, P21-pcDNA3.1-K562 cell clone that stably expression P21(WAF) was isolated. P21(WAF) protein was identified by Western blot. The survival rate were tested by MTT. Cell cycle and apoptosis were tested by flow cytometry. The results showed that the expression of P21(WAF) protein could be detected by Western blot in P21-pcDNa3.1-K562 cells. A strong inhibition of cell proliferation was observed in P21-pcDNA3.1-K562 cells as compared with that of the control. The cells cycle were arrested in G(0)/G(1) phase. The percentage of apoptosis was declined slightly after P21-pcDNA3.1-K562 cells were combined with STI571, meanwhile its survival rate declined more slowly than that of K562 cell with STI571. In conclusion, P21(WAF) inhibits the proliferation of K562 cell, meanwhile slightly inhibits its apoptosis induced by STI571and decrease its sensitivity to STI571.

[Effects of mitogen-activated protein kinase(MAPK) in cell signal transduction of chronic myelogenous leukemia cell line K562].

BACKGROUND & OBJECTIVE: Ras-MAPK signal transduction has been thought to play an important role in the carcinogenesis of chronic myelogenous leukemia. In this study, the authors investigated the effects and mechanism of mitogen-activated protein kinase (MAPK) in cell signal transduction of chronic myelogenous leukemia(CML). METHODS: After MAPK antisense oligodeoxynucleotide (ASO) was introduced into K562 cell line by liposomal transfection, the effects of ASO on K562 cell were evaluated by cell proliferation, DNA synthesis, MAPK protein content and MAPK activity. RESULTS: The cell proliferation, DNA synthesis, MAPK protein content and MAPK activity were significantly inhibited by MAPK ASO, the inhibitory rates were 51.8%, 57.1%, 45.3%, and 61.6%, respectively,with significant difference in comparison to the control (P<0.05). CONCLUSION: MAPK plays an important role in the signal transduction of CML and MAPK may become a new target in the treatment of CML.

[Effect of PD98059 on Ras-MAPK signal transduction pathway of chronic myelogenous leukemia].

AIM: To study the effect and mechanism of PD98059 on Ras-MAPK signal transduction pathway of chronic myelogenous leukemia. METHODS: K562 cell line was treated by PD98059, cell viability, DNA synthesis, colony formation and MAPK activity of the treated cells were analyzed. RESULTS: The cell viability, DNA synthesis, colony formation and MAPK activity were significantly inhibited by PD98059 (P<0.05), and the inhibitory effect was dose dependent. CONCLUSION: The inhibitory effect of PD98059 was achieved by blocking Ras-MAPK signal transduction pathway which can become a new target in the treatment of CML.

[Apoptosis of K562 cells induced by antisense-oligonucleotides of CRKL gene].

BACKGROUND & OBJECTIVE: It was recently reported that the proteins coded by CRKL (CT10 regulator of kinase like, CRK like) gene play an important role in the pathogenesis of chronic myeloid leukemia (CML). This study was designed to investigate the effect of antisense oligonucleotides of CRKL gene (CRKL-ASDON) on CML K562 cell lines. METHODS: K562 cells were transfected with CRKL-ASDON, using liposome as the vector. The changes of cell morphology, cell cycle, and gene expression were observed through living cell count, transmission electron microscopy, flow cytometry (FCM), DNA Ladder assay. RESULTS: Under the action of CRKL-ASDON, growth of K562 cell was markedly inhibited. An apoptotic peak appeared before diploid peak in FCM; various apoptotic stages of K562 cells were observed under electron microscope; over ladder-shaped band of cell apoptosis was seen in abstracted DNA of the gene group by gel electrophoresis. Meanwhile, the above changes did not appear in the control. CONCLUSION: CRKL gene is an important factor in the pathogenesis of Ph-positive CML.