[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.

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 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.