Clinical effect of Tip-Edge plus appliance in children with angle II(1) malocclusion.

The effects of Tip-Edge plus appliance in the treatment of Angle II(1) malocclusion and the mechanism were investigated. Fifty-two Angle II(1) children, aged from 12.3-14.2 years, with mandibular retrusion in permanent dentition were selected and treated with Tip-Edge plus appliance. Lateral cephalometric films taken before and after treatment were analyzed. The arithmetic mean and standard deviation were calculated for each variable. Paired t-test was performed to evaluate the significant treatment change. Results showed that the average treatment time was 16 months. Normal overjet and overbite were established with retroclination of upper incisors and proclination of lower incisors. U1-NA was decreased by 15.4° (P<0.01). ANB and Y axial angle were decreased significantly (P<0.05). Soft tissue measurements showed that FCA and UL-E were decreased dramatically (P<0.05), and LL-E was increased significantly (P<0.05). Remarkable soft tissue change was noted after the treatment and convex facial profile changed to the straight profile. In conclusion, Tip-Edge plus technique can quickly and efficiently correct anterior bite and lateral outlook.

[As2O3 induces demethylation and up-regulates transcription of SHP-1 gene in human lymphoma cell line T2 cells].

OBJECTIVE: To investigate the methylation of CpG island in the SHP-1 gene promoter and its significance in lymphoma. To evaluate the effects of As2O3 on demethylation of SHP-1 in human lymphoma cell line T2 and on proliferation of T2 cells. METHODS: T2 cells were treated with AsO3. Methylation specific PCR was used to detected the status of SHP-1 methylation in newly diagnosed lymphoma tissues and the T2 cells. The mRNA and protein expression of SHP-1 were determined by FQ-PCR and Western blot. The expression of phospha-c-kit was examined by Westren blot. MTT and flow cytometry were used to determine the growth and apoptosis in T2 cells. RESULTS: T2 cells contained completely methylated SHP-1. Furthermore, there was constitutive c-kit phosphorylation. The expression of SHP-1 was recoverd when the cells exposed to AsO3, and concomitant with increasing SHP-1, a parallel down-regulation of phosphorylated c-kit occurred, so that by day 3 phosphorylated c-kit was barely detectable. As2O3 inhibited the cell growth, and the effects were dose- and time-dependent. As2O3 also increased apoptosis rate of T2 cells in a dose- and time-dependent manner, too, and on the 1, 2, 3 d treatment with AsO3 (2.5 micromol/L), the apoptosis rates were 6.12%, 26.53%, 50.90%, respectively. The frequency of methylation in SHP-1 gene promoter in lymphoma tissues was 87.5% (28/32). In the control group, however, 12 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1 gene promoter. CONCLUSION: Hypermethylation of SHP-1 gene promoter in lymphoma indicates the inactivation of SHP-1 gene and its possible role in the tumorigenesis of lymphoma. As2O3 can effectively cause demethylation and inhibit the growth of tumor by reactivating the SHP-1 gene transcription. SHP-1 methylation leading to epigenetic activation of c-kit may have a tentative role in the pathogenesis of lymphoma. Therefore, As2O3 is potentially useful in the treatment of lymphoma as a demethylating agent.

[The mechanism for SHIP gene to induce the apoptosis of human leukemia cell line K562.].

The src homology 2 (SH2)-domain containing inositol-5-phosphatase (SHIP) is another recently identified lipid phosphatase after phosphatase and tensin homology deleted on chromosome ten gene (PTEN). It plays an important role in negatively regulating the proliferation of hematopoietic cells. The relationship between SHIP and the inhibition of tumor proliferation is rarely reported. The purpose of this study is to evaluate the apoptosis induced by SHIP gene in K562 cell line and to explore the involved signaling pathway. The K562 cells were transfected with human SHIP gene by using the lentiviral vector containing SHIP, and the transfection was verified by fluorescent quantitative PCR (FQ-PCR) and Western blot. Then the effects of SHIP protein expression on cell growth and apoptosis were measured. The levels of p-Akt, bcl-2 family, caspase and the activity of NFkappaB were assayed by Western blot and ELISA, respectively. The results are as follows: (1) Human leukemia cell line K562 was SHIP-negative; (2) Transfection with SHIP gene led to the re-expression of SHIP mRNA and protein in K562, as shown by FQ-PCR and Western blot; (3) The expression of SHIP protein inhibited cell growth and significantly increased apoptosis in K562 cells; (4) Compared to that in control group, the expression level of p-Akt-308 and p-Akt-473 in SHIP-expressing cell group decreased significantly (P<0.01); SHIP activated caspase-9, caspase-3, up-regulated protein levels of bad, p27, down-regulated expression of bcl-xL, while it had no effect on the expression of bcl-2 and bax. Furthermore, the inhibition of NF-kappaB was achieved along with the inactivation of Akt. These data suggest that SHIP gene has potential abilities to inhibit K562 leukemic cell proliferation and induce its apoptosis via inactivating PI3K/Akt pathway. The loss of SHIP might be the explanation of aberrant high-level p-Akt in human leukemia. It may be at least one of the mechanisms by which the loss of SHIP expression contributes to leukemia progression.

Role of CXCL12 in metastasis of human ovarian cancer.

BACKGROUND: In a previous study, we have verified that CXCR4 expression is correlated with tumor aggressive progression and poor prognosis in patients with epithelial ovarian cancer. The aim of this study was to explore the effect of CXCL12-CXCR4 axis on the metastasis of human ovarian cancer. METHODS: The expressions of CXCR4 and CXCL12 mRNA and protein in human ovarian cancer cell line CAOV-3 was detected by RT-PCR and immunocytochemistry. Methythiazolyltetrazolium (MTT) was used to analyze the effect of different concentrations of CXCL12 on the proliferation of CAOV-3 cells. Transwell invasion chamber and matrigel were used to evaluate the effect of various concentrations of CXCL12 and ascites on the migration and invasion of CAOV-3 cells. The expressions of integrin beta(1) and vascular endothelial growth factor-C (VEGF-C) mRNA were detected by RT-PCR. Data were analyzed using ANOVA by SAS 6.12. RESULTS: Under serum-free suboptimal culture conditions, CXCL12 (100 ng/ml) significantly enhanced the proliferation of CAOV-3 cells compared with the control and 10 ng/ml CXCL12 groups (0.428 +/- 0.051 vs. 0.325 +/- 0.045 and 0.328 +/- 0.039, P < 0.05). This enhancing effect of CXCL12 was significantly inhibited by 10 microg/ml neutralizing CXCR4 antibody or 1 microg/ml CXCR4 antagonist AMD3100. However, 10 microg/ml neutralizing CXCR4 antibody could not inhibit cell proliferation without CXCL12. The levels of migration and invasion of the CAOV-3 cells treated with 100 ng/ml CXCL12 were significantly higher than those in the control (migration: 523.3 +/- 25.2 vs 108.0 +/- 7.2; invasion: 39.3 +/- 4.0 vs. 4.0 +/- 1.0). The enhancing effect of CXCL12 on cell migration and invasion increased with the concentration of CXCL12 (100 ng/ml vs10 ng/ml: migration, 523.3 +/- 25.2 vs 211.7 +/- 24.7; invasion, 39.3 +/- 4.0 vs 15.7 +/- 3.1, P < 0.05), and was strongly inhibited by 10 microg/ml neutralizing CXCR4 antibody or 1 microg/ml AMD3100. The number of migrated and invading cells in the CAOV-3 added with ascites was significantly higher than those in the 100 ng/ml CXCL12 group (migration: 706.6 +/- 30.6 vs 523.3 +/- 25.2, invasion: 61.7 +/- 7.6 vs 39.3 +/- 4.0, P < 0.05). The level of integrin beta(1) mRNA was greatly increased at 3 hours after being treated with CXCL12 (0.53 +/- 0.10 vs. 1.53 +/- 0.16, P < 0.05), and VEGF-C mRNA displayed significant augment at 24 hours after being treated with CXCL12 (0.52 +/- 0.09 vs 1.11 +/- 0.15, P < 0.05). CONCLUSIONS: CXCL12 and its receptor CXCR4 can promote the proliferation, migration, invasion of ovarian cancer cell line CAOV-3 and enhance its secretion of integrin beta(1) and VEGF-C. These effects can be inhibited by neutralizing CXCR4 antibody or AMD3100. CXCL12-CXCR4 axis plays an important role in ovarian cancer growth and metastasis.

[Effect of chemokine CXCL12 and its receptor CXCR4 on proliferation, migration and invasion of epithelial ovarian cancer cells].

OBJECTIVE: To explore the effect of chemokine CXCL12 and its receptor CXCR4 on proliferation, migration and invasion of epithelial ovarian cancer cells. METHODS: CXCR4 and CXCL12 mRNA and protein expression of human ovarian cancer cell line CAOV3 was detected by RT-PCR and immunocytochemistry. Integrin beta1 and vascular endothelial growth factor-C (VEGF-C) mRNA expression were detected in CAOV3 cells stimulated by CXCL12. The CAOV3 cells were divided into 6 groups: control group (un-stimulated), experimental group 1 (stimulated by 100 ng/ml CXCL12), experimental group 2 (stimulated by 10 ng/ml CXCL12), experimental group 3 (100 ng/ml CXCL12 and 10 microg/ml neutralizing CXCR4 antibody), experimental group 4 (100 ng/ml CXCL12 and 1 microg/ml CXCR4 antagonist AMD3100), experimental group 5 (10 microg/ml neutralizing CXCR4 antibody or ascites). Methyl thiazolyl tetrazolium (MTT) was used to analyze the effects of different concentrations of CXCL12 on CAOV3 cell proliferation. Transwell invasion chamber and reconstructed basement membrane (Matrigel) were used to evaluate effect of various concentrations of CXCL12 and ascites on CAOV3 cell migration and invasion. RESULTS: CAOV3 cells expressed CXCR4 mRNA (0.70 +/- 0.10) and protein, but did not express CXCL12 mRNA or protein. Immunostaining of CXCR4 was mainly located in cytoplasm. CXCR4 mRNA was up-regulated after 100 ng/ml CXCL12 stimulation (1.24 +/- 0.14; t = -7.1088, P = 0.0021). Integrin beta1 mRNA was greatly increased at 3 hours by stimulation of 100 ng/ml CXCL12 (before and after stimulation 0.53 +/- 0.10, 1.53 +/- 0.16; P < 0.01), and VEGF-C mRNA showed significant increase at 24 hours by treatment with CXCL12 (before and after stimulation 0.52 +/- 0.09, 1.11 +/- 0.15; P < 0.05). Under serum-free sub-optimal culture conditions, experimental group 1 greatly enhanced cell proliferation in CAOV3 cells compared with control group and experimental group 2 (respectively 0.428 +/- 0.051, 0.325 +/- 0.045, 0.328 +/- 0.039; P < 0.05). Experimental group 1 was strongly inhibited compared with experimental groups 3 and 4 (the latter two groups respectively 0.356 +/- 0.031, 0.373 +/- 0.029; P < 0.05). There was no significant difference between experimental group 5 (0.349 +/- 0.038) and control group (P > 0.05). Experimental group 1 stimulated the migration and invasion of CAOV3 cells in chemotaxis assay compared with control group and experimental group 2 (number of cell migration respectively 523.3 +/- 25.2, 108.0 +/- 7.2, 211.7 +/- 24.7, number of cell invasion respectively 39.3 +/- 4.0, 4.0 +/- 1.0, 15.7 +/- 3.1; P < 0.01). This enhancing effect of experimental group 1 was strongly inhibited compared with experimental groups 4 and 5 (P < 0.05). The number of migrating and invading cells in experimental group 5 (migration: 706.6 +/- 30.6, invasion: 61.7 +/- 7.6) was significantly higher than that of experimental group 1 (P < 0.05). CONCLUSIONS: This in vitro study shows CXCL12 promote proliferation, migration, invasion of ovarian cancer cell line CAOV3, and up-regulate integrin beta1 and VEGF-C expression, and these effects are strongly inhibited by neutralizing CXCR4 antibody. It suggests CXCL12 and its receptor CXCR4 may play important roles in ovarian cancer growth and metastasis.

[The expression and clinical significance of hematopoietic cell phosphatase and cysteinyl aspartate-specific proteinase in leukemia].

OBJECTIVE: To evaluate the relationship between the expression of hematopoietic cell phosphatase (SHP-1) gene or cysteinyl aspartate-specific proteinases (Caspase-1, 3) gene and the effect of chemotherapy in leukemia patients. METHODS: The expression of SHP-1 and Caspase-1, 3 gene was measured in leukemia patients with reverse transcriptase-polymerase chain reaction. RESULTS: The positive rate of SHP-1 and Caspase-1, 3 gene in acute leukemia was 33.3%, 80.0% and 100.0%. The positive rate of SHP-1 in chronic myeloid leukemia (CML)-blastic phase and CML-chronic phase was 0.0%, 37.5%. The first complete remission (CR) rate of SHP-1 and Caspase-1, 3 positive group was 85.0%, 64.3% and 55.8%, which were higher than that of SHP-1 and Caspase-1, 3 negative group (P < 0.05). There was a positive correlation between SHP-1 and Caspase-1, 3. CONCLUSIONS: SHP-1 gene is potentially an anti-oncogene and may play a role through activation of Caspase-1, 3 gene. The expression of SHP-1 and Caspase-1, 3 is associated with remission rate and it is shown that they participate in the prognosis of leukemia. They could be used as indices for evaluating therapeutic efficacy and judging prognosis in leukemia patients.

[Preliminary study on the effect of TRAIL on adhesion and apoptosis of multiple myeloma cell line RPMI8226 and its mechanism].

The present study was purposed to investigate the inhibition effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on growth of RPMI8226 cells and adhesion between RPMI8226 cells and bone marrow stroma cells (BMSC), and to explore its mechanism as well. The inhibition effects of TRAIL on cells growth and adhesion were assayed by MTT; cell apoptosis was detected by Annexin V and PI; expression of genes bax, bcl-2, mcl-1, CARP1, CARP2, XIAP and cFLIP were determined by semi-quantitative RT-PCR; apoptosis-related protein expression was detected by Western blot. The results showed that TRAIL inhibited the proliferation of RPMI8226 cells in dose- and time-dependent manners. TRAIL induced apoptosis in RPMI8226 cells, the expression level of genes bcl-2, mcl-1, CARP1, CARP2, XIAP and cFLIP decreased, while the expression level of Bax increased, but the expression level of caspase-3 and NF-kappaB P65(RelA) proteins decreased. Moreover, TRAIL up-regulated the expression level of adherent molecule CXCR4 in RPMI8226 cells significantly. It is concluded that TRAIL up-regulated the expression level of adherent molecule CXCR4 in RPMI8226 cells significantly, and induced the apoptosis of RPMI8226 cells. Growth inhibition effect of TRAIL on RPMI8226 cells is in dose- and time-dependent manners.

[Inhibitory effect of lentiviral vector-mediated SHIP gene transfection on proliferation of leukemia K562 cells and PI3K/Akt pathway regulation].

BACKGROUND AND OBJECTIVE: The hemopoietic-restricted Src homology 2-containing inositol 5'-phosphatase (SHIP) acts as a negative regulator for the proliferation and survival of hematopoietic cells by hydrolysing the phosphoinositide 3-kinase (PI3K)-generated second messenger, PtdIns(3,4,5)-P3 (PI-3,4,5-P3) to PtdIns(3,4)-P2 (PI-3,4-P2). This study was to investigate the biological function of SHIP gene in pathogenesis of leukemia cells by lentiviral vector-mediated SHIP transfection. METHODS: Ectopic SHIP gene was transfected into leukemia K562 cells by the mediation of lentiviral vector. The mRNA level of SHIP was detected by fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-PCR). The expression of SHIP, Akt, and phosphorylated Akt (p-Akt) was detected by Western blot. The proliferation and morphology of K562 cells before and after SHIP gene transfection were compared. RESULTS: The proliferation of K562 cells was inhibited after transfection: the proliferation inhibition rate was increased from (9.9+/-1.5)% on Day 3 to (40.6+/-2.3)% on Day 5. K562 cells were SHIP-negative but expressed high level of p-Akt which was down-regulated from 0.533 to 0.245 (P<0.01) after SHIP transfection. Apoptotic characteristics were showed in K562 cells after SHIP transfection. The early apoptosis rate was significantly higher in K562-wtSHIP-FIV-G cells than in K562-FIV-G cells and untransfected K562 cells [(38.3+/-4.3)% vs. (8.2+/-0.9)% and (7.7+/-0.8)%, P<0.05]. CONCLUSIONS: SHIP gene can inhibit cell proliferation and promote cell apoptosis via inactivating PI3K/Akt pathway. Loss of SHIP might activate PI3K/Akt pathway and promote the proliferation of K562 cells.

[Effect of As2O3 on demethylation of SHP-1 gene in human lymphoma cell line T2].

BACKGROUND AND OBJECTIVE: Inducing gene demethylation may be a mechanism of arsenic trioxide (As(2)O(3)) in treating hematologic cancers. This study was to investigate the effect of As(2)O(3) on demethylation of SH2-containing phosphatase-1 (SHP-1) in human lymphoma cell line T2 and on the proliferation of T2 cells. METHODS: T2 cells were treated with As(2)O(3) and 5-aza-2'-deoxyoytidine (5-AC) alone or in combination. The methylation of SHP-1 in T2 cells was detected by methylation-specific polymerase chain reaction (MSP). The mRNA and protein expression of SHP-1 were determined by fluorescence quantitative-polymerase chain reaction (FQ-PCR) and Western blot. The expression of p-c-kit was detected by Western blot. Cell proliferation was detected by MTT assay. Cell apoptosis was detected by flow cytometry. RESULTS: As(2)O(3) led to progressive demethylation and re-expression of SHP-1 in T2 cells, as well as down-regulation of phosphorylated c-kit. As(2)O(3) inhibited the proliferation and promoted the apoptosis of T2 cells, and its effects were enhanced along with the increase of concentration and treatment time (p<0.05). The proliferation inhibition rates were significantly lower in As(2)O(3) (2.5 micromol/L) group than in combination (2.5 micromol/L As(2)O(3) plus 2 micromol/L 5-AC) group (9.8% vs. 11.0%on Day 1, 20.3% vs. 36.7% on Day 2, 47.5% vs. 61.0% on Day 3, p<0.05). The apoptosis rates were significantly higher in combination group than in As(2)O(3) group (17.3% vs. 6.1% on Day 1, 37.9% vs. 26.5% on Day 2, 67.9% vs. 50.9% on Day 3, p<0.05). CONCLUSIONS: As(2)O(3) could cause demethylation and re-expression of SHP-1 in T2 cells, and may inhibit proliferation and induce apoptosis of T2 cells via suppressing activation of c-kit receptor and its signal transduction.

[Expression and clinical value of SHP-1 and c-kit in acute leukemia].

The aim of study is to investigate the expression of hematopoietic cell phosphatase (SHP-1) gene and c-kit pro-oncogene in acute leukemia (AL) and its impact on prognosis in AL. Semi-quantity reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of SHP-1 mRNA and c-kit mRNA in 60 AL patients and 33 normal controls (NC). The results showed that the positive rates of SHP-1 expression from high to low level were found orderly in complete remission group, newly diagnosed group and relapsed group, there was significance difference between each group and NC group (P < 0.05). The positive rates of c-kit expression were opposite order in each groups as compared with SHP-1. there was also significance difference between each group and NC group (P < 0.05). The positive rate of SHP-1 and c-kit expressions in AML was higher than that in ALL (P < 0.05), there was negative correlation between expressions of SHP-1 and c-kit (r = -0.502, P < 0.05); The difference between the complete remission rate in SHP-1 positive and in SHP-1 negative patients from 30 newly diagnosed AML patients was significant (P < 0.05), the same result was found between c-kit(+) complete remission and c-kit(-) complete remission. It is concluded that SHP-1 gene is a potentially anti-oncogene and inhibits the growing of tumor by negatively modulating c-kit gene. Simultaneous detection of SHP-1 and c-kit gene may act as a factor for predicting prognosis in AL.

[Combined effect of recombinant mutant human TRAIL and daunorubicin in inducing apoptosis of leukemia cell and its mechanism].

The aim of study was to investigate the combined effect of recombinant mutant human TRAIL (rmhTRAIL) with daunorubicin (DNR) or alone on K562 and U937 leukemia cell lines and its mechanism. The fibroblasts (MRC-5) of normal-human embryonic lung were used as control cells. After being treated with rmhTRAIL and DNR or only with rmTRAIL, the cytotoxic effect and the apoptosis rate in K562, U937 cells were measured by MTT assay. The expression levels of TRAIL death receptor and TRAIL decoy receptor mRNA in these three cell lines were assayed by semiquantitive RT-PCR before and after treatment with DNR. The results indicated that K562 and U937 were sensitive to rmhTRIAL. DNR had synergistic inhibitory effect with rmhTRAIL on the growth of K562 and U937 cell lines (P < 0.05). The expression level of DR4 and DR5 mRNA was significantly higher in K562 and U937 with combined treatment of rmhTRAIL and DNR than that in those alone, while the expressions of DcR1 and DcR2 mRNA were not influenced. It is concluded that in vitro, rmhTRAIL alone or in combination with DNR can obviously inhibit the growth of leukemia cell lines and induce cell apoptosis, DNR and rmhTRAIL have a synergistic inhibitory effect on growth of K562 and U937. The mechanism may correlate with the up-regulation of DR4 and DR5 of K562 and U937.

[Clinic Significance of Expression of bcl-2 and bax Gene in Patients with Acute Leukemia and its Relationship with mdr-1 Gene Expression]

It is generally accepted that the inhibition of apoptosis is one of the mechanism of drug resistance to tumor. Members of the bcl-2 gene family are the most important regulators in apoptosis. The purpose of this study is to evaluate the value of expression of bcl-2 and bax gene in predicting the prognosis of acute leukemia patients, and to explore the relationship between bcl-2 and bax expression and drug resistance. Seventy patients with acute leukemia entered this study. Expressions of bcl-2, bax and mdr-1 gene were measured by RT-PCR method and FCM. The result showed that: bcl-2 had been widely detected in specimens of blood or bone marrow from acute leukemia patients, the expression levels were much higher than those in normal control (1.46 vs 0.71, P < 0.05), bax expression levels and bax/bcl-2 ratio in patients had no significant difference with the control. No relationships were found between the expression levels of bcl-2 and bax and AL patients' age, sex, platelet counts, hemoglobin levels, percentage of marrow blasts, FAB classification, and S + G(2)M%. Both Bcl-2 protein expression (34.6% vs 69.2%, P < 0.03) and bax/bcl-2 mRNA ratio (37.1% vs 82.9%, P < 0.01) were associated with response to therapy and CR rate, bax/bcl-2 ratio also influences the overall survival time. There was no relationship between bcl-2 and bax expression levels and mdr-1 expression levels.

[Effects of sodium orthovanadate on proliferation and apoptosis in raji cells and its mechanism].

In order to investigate the role and the mechanism of protein tyrosine phosphatase (PTPase) signaling pathway in the regulation of proliferation, cell cycle and apoptosis in lymphoma cells, the effects of sodium orthovanadate, Na(3)VO(4), a specific PTPase inhibitor, were explored on Raji lymphoblast-like cell line by MTT assay and CFU-Raji culture, morphologic observation, DNA gel electrophoresis, FCM and RT-PCR. Results showed that MTT assay and CFU-Raji culture demonstrated that sodium or thovanadate inhibited the growth of Raji cells in a concentration-dependent fashion; morphologic observations showed that Raji cells exhibited cytoplasm shrinkage, cytoplasm membrane blebbing, nuclear fragmentation and chromatin condensation forming crescents along nuclear membrane characteristic of apoptosis in the presence of Na(3)VO(4); DNA gel electrophoresis revealed typical DNA ladder reminiscent of DNA cleavage at internucleosomal sites in Na(3)VO(4) treated cells; FCM and RT-PCR indicated that Na(3)VO(4) intervention increased the fraction of annexin V(+) PI(-) cells, reduced the value of mitochondrial transmembrane potential, induced G(2)/M arrest and down-regulated the expression of Bcl-2 and cyclin B1 at both mRNA and protein level in a concentration-dependent manner. It was concluded that PTPase pathway might be implicated in the regulation of cell proliferation, cell cycle and apoptosis, and PTPase specific inhibitor Na(3)VO(4) could induce Raji cell growth inhibition, G(2)/M arrest and apoptosis via down-regulation of Bcl-2 and cyclin B1, and reduction of mitochondrial transmembrane potential.

[In vitro effects of mevastatin on the proliferation and apoptosis in human multiple myeloma cell line U266].

In order to investigate the anti-tumor activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors and the mechanism underlying the cell proliferation and apoptosis modulated in myeloma cells, the effects of mevastatin, an HMG-CoA reductase inhibitor, on cell growth, cell cycle progression and apoptosis in U266 human multiple myeloma (MM) cell line in vitro were explored by MTT colorimetric assay, morphologic observation, flow cytometry, DNA gel electrophoresis, and RT-PCR. The results demonstrated that mevastatin inhibited the growth of U266 cells in time- and dose-dependent manners. Cell cycle analysis showed that U266 cells underwent G(0)/G(1) arrest under exposure to mevastatin, but it did not affect p27 expression at both mRNA and protein level. Morphologic observations revealed cytoplasm shrinkage, nuclear condensation and fragmentation in mevastatin-treated cells, and fraction of annexin V(+)PI(-) cells increased significantly in the presence of the agent as determined by flow cytometric assay. In addition, mevastatin caused the collapse of mitochondrial transmembrane potential (Deltapsim), induced DNA fragmentation, and down-regulated the mRNA expression of bcl-2. The growth-inhibitory, cell cycle arresting, and proapoptotic effects of mevastatin in U266 cells could be effectively reversed by the addition of mevalonate (MVA), the immediate endproduct of the reaction catalyzed by HMG-CoA reductase. It is concluded that mevastatin suppresses proliferation by inducing G(0)/G(1) phase arrest and triggering apoptosis via down-regulation of bcl-2 and reduction of Deltapsim, which may be attributed to the inhibition of MVA pathway by mevastatin. Statins including mevastatin may find their future application in the treatment of MM.

[Effects of bcl-2 antisense oligodeoxynucleotide on proliferation and apoptosis of Raji cells].

OBJECTIVE: To study the in vitro antitumor activity of bcl-2 fully phosporothioated antisense oligodeoxynucleotide (bcl-2 ASODN) to malignant lymphoblastic cells. METHODS: Proliferation and apoptosis of Raji cells incubated with bcl-2 ASODN were evaluated by MTT assay, flow cytometry (FCM) and electron microscopy, and the level of bcl-2 protein and mRNA expression were assessed by FCM and RT-PCR, respectively. RESULTS: MTT assay demonstrated that bcl-2 ASODN could partially inhibit the growth of Raji cells. After incubated with ASODN for 48 hours, Raji cells exhibited characteristic morphologic changes of apoptosis, including cytoplasm membrane blebbing, chromatin condensation crescents formation and nuclear fragmentation. The apoptosis rate of Raji cells treated with 20 micromol/L bcl-2 ASON for 72 hrs was 43.86% which is significantly higher than that of control (10.05%). The bcl-2 ASODN induced apoptosis of Raji cells was accompanied by declined expression of bcl-2 mRNA, which decreased to 0.88% at 72 hrs and was significantly lower than that of control (79.54%). CONCLUSION: bcl-2 ASODN induced Raji cells apoptosis by downregulating bcl-2 protein.

[Effects of aminophylline on proliferation and apoptosis in Raji lympho-blastoid cell line].

The aim of this study was to investigate whether and how phosphodiesterase (PDE) inhibitors modulate the proliferation, cell cycle and apoptosis in lymphoma cells. The effects of aminophylline (AM), a non-specific PDE inhibitor, on Raji cells were explored in vitro. MTT assay, light and transmission electron microscopy and annexin V staining were used to observe cell proliferation, morphologic changes and apoptosis rate in AM-treated cells, and FCM and RT-PCR techniques were adopted to detect the effect on cell cycle, the expression of cyclin B1 and Bcl-2 and mitochondrial transmembrane potential in AM-treated cells. The results showed that AM inhibited the growth of Raji cells in a concentration-dependent manner. Morphologic observations showed apoptosis changes in AM-treated cells, including cytoplamic shrinkage, cytoplasmic bubbling, karyopyknosis and nuclear fragmentation. FCM and RT-PCR detection showed that AM intervention increased the fraction of annexin V(+) cells, reduced the value of mitochondrial transmembrane potential, induced S phase arrest, and down-regulated the expression of Bcl-2 at both mRNA and protein level and cyclin B1 protein in a concentration-dependent manner. It is concluded that PDE inhibitor aminophylline may induce Raji cell growth inhibition, S phase arrest, apoptosis via down-regulation of Bcl-2 and reduction of mitochondrial transmembrane potential.

[Expression of survivin, Fas, bcl-2 and bax in bone marrow cells from acute myeloid leukemia patients and its clinical significance].

UNLABELLED: To study the clinical significance of the expression of antiapoptosis gene, survivin and bcl-2, and proapoptosis gene, Fas and bax, in acute myeloid leukemia (AML), RT-PCR was used to examine the expression of survivin and flow cytometry (FCM) to detect the expression of Fas, bcl-2, bax and bcl-2/bax ratio in 68 cases of AML. The results demonstrated that: (1) The positivity of survivin mRNA expression was significantly higher in AML compared to control (70.6% vs 30%, P < 0.05). (2) The expression of Fas and bcl-2 in AML before treatment was significantly higher than that in control (P < 0.001), but the bax expression did not (P > 0.05). (3) The survivin-positive AML cases showed a significantly lower Fas and higher bcl-2 expression in comparison with survivin-negative ones (P < 0.01 and P < 0.001, respectively), but the bax did not (P > 0.01). (4) Survivin-positive AML cases had a lower CR rate as compared with survivin-negative cases (64.6% vs 90%, P < 0.05). (5) The survivin-positive CR cases showed a decreased expression of Fas and bcl-2 after treatment in comparison with pretreatment expression (P < 0.001), but the bax expression remained unchanged before and after therapy. The survivin-positive NR cases showed a significantly decreased Fas and increased bcl-2 expression as compared with pretreatment expression (P < 0.001). bcl-2/bax ratio was also significantly higher in NR cases. IN CONCLUSION: 70.6% AML cases showed positive for survivin expression with a lower CR rate, the survivin-positive AML showed a low Fas with high bcl-2 expression and bcl-2/bax ratio as compared to the survivin-negative cases.

[Improved RT-PCR for detection of PML/RARalpha fusion gene in rapid diagnosis of acute promyelocytic leukemia].

Detection of the PML/RARalpha fusion gene by RT-PCR in acute promyelocytic leukemia (APL) blasts is not only critical to commence promptly the specific therapy with all-trans retinoic acid (ATRA) or arsenic trioxide (As(2)O(3)), but also essential for the definition of PML breakpoint type and subsequent monitoring of minimal residual disease (MRD). The current PML/RARalpha amplification techniques with conventional nested PCR are laborious and time consuming, which fails to meet the requirements for rapid diagnosis of APL in clinical practice. Therefore, an easily handled RT-PCR methodology for the rapid and accurate amplification of PML/RARalpha fusion transcripts is needed. A modified one round RT-PCR protocol was described with a few variations which includes rapid extraction of high quality cellular total RNA, cDNA synthesis with random hexamer and M-MLV reverse transcriptase, optimal concentrations of MgCl(2) (1 mmol/L), PCR primers (0.4 micro mol/L) and Taq polymerase (0.01 U/ micro l), hot-start procedure, and concomitant amplification of PML/RARalpha fusion gene and RARalpha internal control under the identical thermocycle parameters. The results in 40 patients with newly diagnosed APL showed that the improved RT-PCR protocol allowed the rapid detection of PML/RARalpha fusion gene and the accurate discrimination of its transcript types, and simultaneous amplification of RARalpha internal control under the identical program in less than 6 hours. There were no false positive or negative results found with the assay. In conclusion, the assay reported here is proved to be a simple, easily handled, and highly specific procedure for the diagnosis of APL cases, particularly those requiring such urgent therapeutic intervention as ATRA or As(2)O(3) and meriting its further application in APL management.

[Relationship between resistance to chemotherapy and expression of breast cancer resistance protein (BCRP) gene in patients with acute leukemia].

In order to investigate the relationship between the expression of breast cancer resistance protein (BCRP) gene and drug resistance in patients with acute leukemia (AL), semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of BCRP mRNA in AL patients and 15 normal subjects. Beta(2)-microglobin (beta(2)-MG) was used as positive reference. BCRP/beta(2)-MG ratio >or= 0.5 was defined as BCRP mRNA positive. The results showed that the positive percentage of BCRP gene expression in newly diagnosed group was 37.6%. The first complete remission rate was 79.3% and 31.6% in BCRP mRNA negative and BCRP mRNA positive patients respectively. The difference was significant (P = 0.001). The expression levels of BCRP mRNA in drug resistance group and drug sensitive group were 0.962 +/- 0.426 and 0.315 +/- 0.296 respectively (P = 0.0001). The expression level of BCRP mRNA in relapsed/refractory group was significantly higher than that in newly diagnosed group (P = 0.0025). The expression level of BCRP gene in normal individuals and long-term survival group was very low and correlated with FAB subtypes. It is concluded that high expression of BCRP mRNA leads to clinical drug resistance and is an unfavorable factor for prognosis of AL patients except acute promyelocytic leukemia.