[Role of P27(Kip1) and TGF-beta1 in APL cell apoptosis induced by As(2)O(3)].

The aim of study was to investigate the effects of arsenic trioxide (As(2)O(3)) on cell cycle and apoptosis of APL cells, as well as changes of P27(Kip1), endogenous TGF-beta1, cyclin E and bcl-2, and to explore the relationship between expression of P27(Kip1) and apoptosis induced by As(2)O(3). The apoptosis and cell cycle changes of APL cells treated with As(2)O(3) were detected by morphology and flow cytometry respectively, the protein and mRNA expressions of P27(Kip1), TGF-beta1, cyclin E and BCL-2 were measured by immunohistochemistry and RT-PCR. The results indicated that As(2)O(3) induced APL cell apoptosis in vitro, and cell cycle was arrested at G(1) phase. Apoptotic cells induced by As(2)O(3) 1, 5 and 10 micromol/L for 24 hours were 1.42%, 4.57% and 10.67% respectively; the proportion of apoptotic cells induced by As(2)O(3) of same concentrations for 48 hours increased to 8.92%, 16.07% and 18.90% respectively; the cells induced by As(2)O(3) for 72 hours were mainly in debris. Protein and mRNA expressions of P27(Kip1) and TGF-beta1 of APL cells after treatment with As(2)O(3) increased, accompanying with decrease of cyclin E, bcl-2 protein and mRNA expressions. Apoptotic cells were related to the expressions of P27(Kip1) (r(mRNA) = 0.55, p < 0.05) and TGF-beta1 (r(mRNA) = 0.51, p < 0.05). There was positive correlation between the expression of TGF-beta1 and of P27(Kip1) (r(mRNA) = 0.31, p < 0.05). It is concluded that the apoptosis of APL cells is induced by As(2)O(3), and the cell cycle is arrested at G(1) phase. The expression of P27(Kip1) is closely related to the extent of apoptosis induced by As(2)O(3). Apoptosis of APL cells induced by As(2)O(3) may be caused by up-regulating TGF-beta1 and P27(Kip1), which is antagonistic to cyclin E and BLC-2, leading to arrest of cell cycle at G(1) phase.

[Rearrangements of the mixed lineage leukemia gene in acute myeloid leukemia].

OBJECTIVE: To study the frequency of mixed lineage leukemia (MLL) gene rearrangements in patients with acute myeloid leukemia (AML) and to determine the significance thereof. METHODS: Conventional cytogenetics (CC) and karyotype analysis were conducted on the bone marrow cells from 58 patients with acute myelocytic leukemia (AML), 47 adults (aged 15 approximately 67) and 11 children (aged 1 approximately 14). Fluorescence in situ hybridization (FISH) using the whole chromosome painting (WCP) probes of the chromosomes 1, 5, 11, 16, 17, and 21 was performed. A total of 58 patients were included in this study. Forty-seven of these patients with AML were adults and the remaining were children. Both conventional cytogenetics (CC) and fluorescence in situ hybridization (FISH) were carried out. FISH analysis was performed utilizing commercially available DNA probes, including whole chromosome painting probes, locus specific probes, and specific and dual color/multiple color translocation fusion probes. RESULTS: Six out of the 58 patients (10.3%) were found to have MLL gene rearrangements, either an extra signal of MLL gene or a disruption of MLL gene due to a translocation or deletion or duplication. Of these six patients with MLL gene rearrangements, four were adults and two were children. In addition to MLL gene rearrangements, complex chromosomal changes were also detected in five of these patients: 47 - 49, XX, der (1) t (1; 17) (p36.1; q23), +4, +10, der (11) t (11; 17) (q23; q23), -17, -18, +20, +21?. ish +21 (wcp21+), der (1) t (1; 17) (wcp17+), der (11) t (11; 17) (wcp11+; wcp17+); 46, XX, del (5) (q13q33), r (11) (p15q25), +r (11) (p15q25). ishr (11) (wcp11+, MLL+), +r (11) (wcp11+, MLL+); 46, XY, del (11) (q23) [2]/46, idem, add (16) (p13.1) [8]/46, XY [10]. ishadd (16) (wcp16+), rea (11) (wcp11+); 55, XY, + markers, ish 11q23 (MLL x 3), +21 (wcp21+); and 46, XY, add (11) (q23) [6]/46, idem, t (15; 17) (q22; q21) [12]/46, XY [2]. ish dup (11) (MLL++), t (15; 17) (PML+, RARa+; RARa-) [24]. CONCLUSION: MLL gene rearrangement is relatively common in AML patients. Because MLL gene arrangements due to translocation or other structural changes are associated with poor response to chemotherapy and poor prognosis, routine testing for this gene rearrangement should be provided to all newly diagnosed patients with AML.

[Enhancement of all-trans retinoic acid-induced differentiation by bufalin in primary culture of acute promyelocytic leukemia cells].

OBJECTIVE: To investigate the effect of bufalin combined with all-trans retinoic acid-induced (ATRA) differentiation of acute promyelocytic leukemia (APL) cells in primary culture. METHODS: Fresh leukemia cells were obtained from heparinized bone marrow aspirations of 12 newly diagnosed APL patients. Cell viability was determined by trypan blue dye exclusion. Apoptosis of APL cell was assessed by morphological analysis. Differentiation of APL cell was also assessed by morphological analysis. Nitro blue tetrazolium (NBT) reduction test and expression of the granulocyte/macrophage-specific antigen CD(11)b was carried out with flow cytometric assay. RESULTS: Bufalin combined with ATRA can induce differentiation of APL cells towards mature stages, NBT reduction was increased 15% - 52% and CD(11)b expression was also increased 16% - 69% in combination of bufalin and ATRA as compared with that of ATRA alone, while the concentration of ATRA needed in the combination group was reduced to 30% and the time of differentiation was reduced from 7 days to 4 days. CONCLUSION: The combination of ATRA with bufalin can significantly enhance the differentiation of acute promyelocytic leukemia cells in primary culture by ATRA.

[Studies on gene expression and the 5' CpG islands methylation status of E-cadherin in acute myeloid leukemia].

OBJECTIVE: To study the relationship between E-cadherin gene expression and the methylation status of E-cadherin 5' CpG islands in acute myeloid leukemia (AML). METHODS: Reverse transcription-PCR (RT-PCR), flow cytometry and methylation specific PCR were used to analyze the E-cadherin gene and protein expression and its 5' CpG island methylation status respectively in bone marrow cells from 55 AML patients and 7 normal controls. RESULTS: AML cells displayed a significant reduction or lack of E-cadherin gene and protein expression, the positive rates were 23.6% and 18.2% (P < 0.01), respectively. All normal bone marrow cells were E-cadherin positive. Thirty-eight of the 55 patients (69.1%) were E-cadherin 5' CpG island methylated whereas the normal controls were completely unmethylated. Twenty-nine of thirty-one (93.5%) E-cadherin-negative samples showed abnormal hypermethylation of the E-cadherin CpG islands. CONCLUSION: Expression downregulation and methylation of E-cadherin gene in AML suggest that it might be an important event in AML. E-cadherin methylation was associated with the inhibition of E-cadherin gene and protein expression in AML.

[Identification of chromosome 21 anomalies in patients with acute myeloid leukemia by fluorescence in situ hybridization].

OBJECTIVE: To investigate the possible complex anomalies of chromosome 21 in patients with acute myeloid leukemia (AML). METHODS: Fluorescence in situ hybridization (FISH) was performed by using commercially available DNA probes, including whole chromosome painting probes, locus specific probes, and specific and dual color translocation fusion probes, on 50 AML patients, 37 adults and 13 children. RESULTS: Anomalies of chromosome 21 were found in 7 patients (14%), including numerical chromosomal anomalies and structural rearrangements. Four of the 13 pediatric patients were found to have trisomy of chromosome 21, among which one had an additional chromosome rearrangement: 47-49,XX,der(1)t(1;17)(p36.1;q23), +4, +10, der(11)t(11;17)(q23;q23), -17, -18, +20, +21. Three out of the 37 adult patients were found to have structural rearrangement of chromosome 21, I.e., t(8;21), among which one had an additional duplicated derivative chromosome 21 plus duplication 15q:46,XX,der(21)t(8;21), dup(15q). CONCLUSION: Rearrangement of chromosome 21 is common in both childhood and adult patients with AML. However, childhood patients tend to have numerical change of chromosome 21, whereas the adult patients are likely to have structural changes of chromosome 21.

[Analysis of methylation status of the promoter of mdr1 gene in K562 and K562/DNR cells].

OBJECTIVE: To analyze the methylation patterns of mdr1 gene promotor in both K562 cell and K562/DNR cells and the relationship between promotor methylation and P-gp expression. METHODS: mdr1 gene expression was analyzed by flow cytometry and RT-PCR, methylation status of the promotor of mdr1 gene by bisulfite-sequencing (including two GC-box). RESULTS: mdr1 gene was found methylated at GC-box and not expressed in K562 cells, but unmethylated and expressed respectively in K562/DNR cells. CONCLUSION: The methylation patterns of mdr1 gene promotor in K562/DNR and K562 cells were different. mdr1 gene silencing was associated with the promotor methylation.

[Effect of PKC inhibitor on P-gp expression and drug-resistance in MGC803 cells].

BACKGROUND & OBJECTIVE: It is showed that there is close relationship between multidrug resistance (MDR) and protein kinase C signal transduction system, but the mechanism remains unclarified. The aim of this study was to investigate the correlation between protein kinase C (PKC) signal transduction system and mdr1 gene in human gastric cancer cell line through studying the effect of vincristine (VCR) and a selective inhibitor of PKC, myr-psiPKC on MGC803 cells. METHODS: Western blot analysis was used to analyze the expression of P-glycoprotein (P-gp), which was encoded by mdr1, in transient VCR induced MGC803 cells, which were treated with or without myr-psiPKC. Cell cycle analysis was performed using flow cytometry and MTT assay was used to investigate the drug susceptibility of MGC803 cells which were exposed to VCR with or without myr-psiPKC. RESULTS: High level of P-gp expression was detected in the MGC803 cells after transient exposure to VCR, and its expression was down-regulated when the same VCR induced MGC803 cell line was incubated with myr-psiPKC. FCM results indicated that more MGC803 cells showed significantly higher level of apoptotic phenotype when treated with VCR and myr-psiPKC (ratio 31.23%), than those treated with only VCR (ratio 18.42%). The IC(50) (284.0+/-13.2 ng/ml) to VCR of MGC803 cells pretreated with VCR exhibited 2.24-fold of negative control group (127.0+/-17.6 ng/ml) and 1.33-fold of the group (212.0+/-30.4 ng/ml) treated with myr-psiPKC. CONCLUSION: The expression of P-gp can be induced by transient exposure to VCR and this induction can be inhibited by myr-psiPKC, which blocks the activity of PKCalpha and beta. PKC signal transduction system may play certain roles in modulating mdr1 expression in gastric cancer.

Effect of mitogen-activated protein kinase signal transduction pathway on multidrug resistance induced by vincristine in gastric cancer cell line MGC803.

AIM: To investigate the correlation between mitogen-activated protein kinase (MAPK) signal transduction pathway and multidrug resistance (MDR) in MGC803 cells. METHODS: Western blot was used to analyze the expression of MDR associated gene in transient vincristine (VCR) induced MGC803 cells, which were treated with or without the specific inhibitor of MAPK, PD098059. Morphologic analysis of the cells treated by VCR with or without PD098059 was determined by Wright-Giemsa staining. The cell cycle analysis was performed by using flow cytometric assay and the drug sensitivity of MGC803 cells which were exposed to VCR with or without PD098059 was tested by using MTT assay. RESULTS: Transient exposure to VCR induced P-gp but not MRP1 or GST-pi expression in MGC803 cells and the expression of P-gp was inhibited by PD098059. Apoptotic bodies were found in the cells treated with VCR or VCR+PD098059. FCM results indicated that more MGC803 cells showed apoptotic phenotype when treated by VCR and PD098059 (rate: 31.23%) than treated by VCR only (rate: 18.42%) (P<0.05). The IC(50) (284+/-13.2 mug/L) of MGC803 cells pretreated with VCR was 2.24-fold as that of negative control group (127+/-17.6 mug/L) and 1.48-fold as that of the group treated with PD098059 (191+/-27.9 mug/L). CONCLUSION: This study shows that the expression of P-gp can be induced by transient exposure to VCR and this induction can be prevented by PD098059, which can block the activity of MAPK. MAPK signal transduction pathway may play some roles in modulating MDR1 expression in gastric cancer.