Expression of cyclin A1 mRNA in patients with myelodysplastic syndrome and its clinical significance / 中国实验血液学杂志

The purpose of this study was to evaluate the expression of cyclin A1 mRNA in patients with myelodysplastic syndrome (MDS) and its clinical significance. The expression of cyclin A1, cdk2 and p21(cip1) mRNA in the bone marrow from 56 patients with MDS and 10 normal control were measured by using reverse transcription polymerase chain reaction (RT-PCR) technique. The results indicated that the positive rate and the expression level of cyclin A1 in MDS patients (69.64%; 0.964 +/- 1.879) were significantly higher than those in normal control (0%; 0.012 +/- 0.014) (p < 0.01). Among de-novo MDS patients, the expression level of cyclin A1 mRNA in the MDS-RAEB group (1.895 +/- 1.769) was higher than that in MDS-RA group (0.629 +/- 1.583) (p < 0.01). The expression level of cyclin A1 mRNA in post-treatment group was significantly lower than that in prior-treatment group (p < 0.01). It is concluded that the mRNA expression of cyclin A1 in MDS patients is higher than that in normal control, the abnormal expression of cyclin A1 may be used as a prognostic marker in MDS patients.

Expression of midkine in patients with acute myeloid leukemia and its significance / 中国实验血液学杂志

The study was aimed to investigate the expression of midkine (MK) in bone marrow mononuclear cells (BM MNC) from 65 acute myeloid leukemia patients and 15 normal controls. The method of RT-PCR was used to examine the expression of MK mRNA in BM MNC. Parts of samples were incubated for 24 hours and the gene expression of MK in the BM MNC was detected by means of Western blot. The results showed that the expression of MK of BM MNCs in 50 newly diagnosed AML patients (0.331 +/- 0.436) and 15 AML patients in relapse (0.374 +/- 0.463) were markedly higher than that in 15 CR cases (0.067 +/- 0.190), and 15 normal controls (0), respectively. The complete remission in MK positive patients (63.16%) was significantly lower than that in MK negative group (93.55%). The patients with positive MK expression had a higher relapse rate than those with negative MK expression. The positive rate of MK gene expression in drug-resistant patients and drug-sensitive patients were 57.69% and 25.64% respectively and there was positive correlation between the gene expressions of MK and bcl-2 (P < 0.01) (r = 0.0556, P < 0.001). It is concluded that MK can be secreted by AML cells and involved in drug-resistant, its positive expression may be associated with the poor prognosis in newly diagnosed AML patients. The inhibitory effect of MK on apoptosis of leukemic cells is induced by upregulating bcl-2 expression.

Relationship between VEGF and MMP-2, MMP-9 in 82 patients with acute myeloid leukemia / 中国实验血液学杂志

In order to investigate the relationship between VEGF and matrix metalloproteinase (MMP)-2, -9 in acute myeloid leukemia patients, and evaluate the significance of them in extramedullary leukemic invasion, the expressions of MMP-2 mRNA, MMP-9 mRNA, VEGF mRNA in bone marrow from 86 patients with acute myeloid leukemia (AML), as well as human hematopoietic cell lines were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The proteolytic activities of MMP-2 and MMP-9 in the supernatants were measured by zymography. The VEGF protein in serum of all samples was detected by ELISA. All these results were analyzed for determination of the relationship between VEGF and MMP-2, MMP-9. The results showed that there was a positive correlation between expressions of MMP-2 mRNA or MMP-9 mRNA and VEGF mRNA or protein. But no such correlation was demonstrated in the AML (CR) and normal control (NC) groups. A higher expression level of MMP-2 and MMP-9 in the VEGF positive group was found, as compared with the negative group (P < 0.05). More extramedullary infiltration occurred in VEGF positive groups than that in VEGF negative groups of AML. The expression of bcl-2 in HL-60 cells was upregulated by VEGF. It is concluded that there are significantly positive correlations between the expression of MMP-2 and MMP-9 with VEGF mRNA or protein levels in AML patients. VEGF can upregulate the expression of MMP-2, MMP-9 in HL-60 and a part of the primary leukemic cells. VEGF and MMP-2, MMP-9 may participate in the extramedullary leukemic invasion of AML patients.

Expressions of cyclin E2 and survivin in acute leukemia and their correlation / 中国实验血液学杂志

Cyclin E2 is present in solid tumors, while its expression and clinical value in acute leukemia is unknown. This study was aimed to investigate the expression of cyclin E2 and survivin gene in bone marrow cells from patients with acute leukemia and their relationship. Reverse transcription polymerase chain reaction was used for detection of the expression of cyclin E2 and survivin mRNA in 84 adult patients with acute leukemia which included 16 cases of relapse, 60 cases of de novo acute leukemia, 8 cases of continuously complete remission, and 20 normal persons as controls. The results showed that (1) positive expression of cyclin E2 (70.24%) in acute leukemia patients was significantly higher than that (0%) in controls, positive expression of survivin (72.62%) in acute leukemia patients was higher than that (30%) in control. (2) the expression of cyclin E2 positively correlated with that of survivin in acute leukemia patients. (3) remission rate in cyclin E2-positive patients (55.81%) was lower than that (88.24%) in cyclin E2-negative patients, the rate of cyclin E2 expression in relapse group was the highest among the three groups; while that in continuously complete remission group was the lowest among the three groups. (4) positive rate of cyclin E2 expression (59.32%) in patients with acute myelocytic leukemia was lower than that (96%) in patients with acute lymphocytic leukemia, no correlation between cyclin E2 expression and white blood cell counts of patients was found. It is concluded that the overexpression of cyclin E2 has been confirmed for the first time to positively correlate with the expression of the survivin in acute leukemia patients, and implicate the poor prognosis. Cyclin E2 may be used as a marker for examination of minimal residual disease.

Expressions of cyclin B1 and p21cipl in adult acute leukemia and their correlation / 中国实验血液学杂志

Cyclin B1, a positive regulator, controls mitosis occurrence, plays an important role in cell proliferation. To investigate the clinical significance of cyclin B1, the expression of cyclin B1 in acute leukemia (AL) patients was measured; the expression of cyclin B1 and p21(cipl), and their cell cycle distribution were assayed by flow cytometry in 136 adult patients with newly diagnosed AL, 10 continuous complete remission (CCR) AL and 17 normal controls; the mRNA of cyclin B1 and p21(cipl), and the proliferation cell nuclear antigen (PCNA) in patients and normal controls were detected with semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The results showed that the expression of cyclin B1 in newly diagnosed AL patients was significantly higher than that in normal controls. For the relapsed AL patients, the cyclin B1 expression was also higher than that in normal controls, but lower than that in newly diagnosed cases, there was no significant difference between the remission cases and normal controls, nor difference between CCR AL patients and normal controls. All patients with high cyclin B1 expression had an unscheduled expression manner, that cyclin B1 protein appeared in G(1) phase, and in some case it even higher than that of G(2) phase. The response rate (partial remission + complete remission) and survival rate in the cyclin B1 high expressed patients were higher than that of cyclin B1 low expressed patients. The relapse rate in cyclin B1 high expressed patients was higher than that in cyclin B1 normally expressed patients. The survival rate in cyclin B1 high expressed patients was higher than that in cyclin B1 low expression patients. A negative correlation between the expression of cyclin B1 and p21(cipl) was observed. Additionally, cyclin B1 protein expression was generally correlated with proliferation index (PI) and proliferation cell nuclear antigen (PCNA). It is concluded that this study demonstrates for the first time cyclin B1 overexpression and abnormally distribution in cell cyclin of newly diagnosed AL patients. It was considered that cyclin B1 may play an important role in leukemic pathogeneses and can be one of the factors influencing the prognosis of AL patients.

Comparative study of expressions of cytoplasmic CD79a and other B-lymphoid immunomarkers in acute leukemic cells / 中国实验血液学杂志

To evaluate the expression of cytoplasmic CD79a (CyCD79a) and other commonly used B-lymphoid immunomarkers including cytoplasmic CD22 (CyCD22), CD19, CD20 and CD10 in various acute leukemia cells and to define the most sensitive and specific markers in the diagnosis of precursor B-cell acute lymphoblastic leukemia (pB-ALL), the immunophenotypic data from 221 de novo adult and pediatric acute leukemia patients as studied using multi-parameter flow cytometry in addition to routine morphologic and enzyme cytochemical assay, were retrospectively analyzed. Cytogenetic and/or molecular biological data in all 45 cases of acute promyelocytic leukemia (APL) and 13 cases of acute leukemia suspected as AML with the fusion genes such as AML1/ETO and CBFbeta/MYH11 were investigated. The results showed that CyCD79a and CyCD22 were the most sensitive and specific markers respectively for pB-ALL. Expression of CyCD79a was seen in 100% of 58 cases of pB-ALL. At the same time, none (0%) of all 147 cases of acute myeloid leukemia (AML) and 15 cases of precursor T-cell acute leukemia (pT-ALL) was positive for CyCD22. The conclusion is made that united detection of CyCD79a and CyCD22 is the optimal immune combination for the diagnosis pB-ALL and the distinguishing pB-ALL with AML and pT-ALL.

Expression of cyclin g2 mRNA in patients with acute leukemia and its clinical significance / 中国实验血液学杂志

To evaluate the expression of cyclin G2 mRNA in patients with acute leukaemia (AL) and its clinical value, the expression of cyclin G2, G1 and P53 mRNA in the bone marrow from 74 AL patients and 10 normal individuals as control were detected with reverse transcription polymerase chain reaction (RT-PCR). The positive segment of cyclin G2 was analyzed by DNA sequencing. The results showed that (1) the positive rate and the expressing level of cyclin G2 in AL patients (52.7%, 0.552 +/- 0.498) were significantly lower than those in normal control (100%, 1.953 +/- 0.675) (P < 0.01); (2) among new diagnosed AL patients, the complete remission (CR) rate (69.2%) in the positive cyclin G2 patients was higher than that (40%) in negative cyclin G2 patients (P < 0.05); (3) the positive rate of cyclin G2 (43.6%) in resistance group was significantly higher than that (68.6%) in sensitive group (P < 0.01); (4) following-up for 14.3 month (11 - 18.5 month) in 28 AL patients with CR, there were 10 relapsed in 11 AL patients with low expression level of cyclin G2 (90.9%); and 7 relapsed in 17 AL patients with high expression (41.2%), and there was significant difference (P < 0.05). In conclusion, the expression of cyclin G2 in AL patients was higher than that in normal control, the abnormal expression of cyclin G2 might be a prognostic marker of CR in AL patients.

Clinical significance of the expression of DNA methyltransferase genes (DNMT) in acute leukemia patients / 中国实验血液学杂志

To investigate the relationship between the expression of DNMT and clinical prognosis in adult patients with acute leukemia (AL), the mRNA expressions of DNMT, p15(INK4B), mdr1 were measured in 72 AL patients and 20 normal controls by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR); the ratio of p15 CpG land methylation was measured in 56 AL patients and 14 normal controls by methylation-specific PCR (MSP-PCR). The results showed that all three DNMT mRNA expressions in AL patients were significantly higher than that in normal controls (P < 0.01). When the internal control was changed into PCNA, a kind of cell proliferation marker gene, the difference still showed a statistic significance. All three DNMT genes were significantly expressed and positively correlated with AL patients, showing high synergistic expression, and there was a negative correlation between the levels of p15, mdr1 gene expression and DNMT. The complete remission (CR) rate in AL patients with the positive expression of all DNMT genes was significantly higher than that of AL patients with partially positive or negative expression (P < 0.01) of DNMT genes. In 56 AL patients, the P15I(NK4B) was completely methylated in 55.4% (31 of 56), partly methylated in 21.4% (12 of 56) and all 14 cases of normal controls were not methylated. It is concluded that DNMT genes are abnormally high expressed in adult AL patients, which lead to methylation-silence of tumor suppressor genes by CpG land hypermethylation, the AL patients with high expression of DNMT are more sensitive to chemotherapy, which may be a good prognostic factor for AL patients.

Cyclin D2 expression in chronic myelogenous leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between cyclin D2 and P210(BCR/ABL) tyrosine kinase in chronic myelogenous leukemia (CML).</p><p><b>METHODS</b>RT-PCR, Western blot and flow cytometry were performed to detect the expression of cyclin D2 in K562 cells and in K562-ib-eGFP cells which express intracellular single-chain antibody (sFv, intrabody) against ABL tyrosine kinase domain.</p><p><b>RESULTS</b>Cyclin D2 expression in K562-ib-eGFP cells was 18.90% which was lower than that of control K562 cells (48.10%), and the number of S-phase cells in K562-ib-eGFP was 40.40% which was much lower than that in K562 cells (64.34%).</p><p><b>CONCLUSION</b>Cyclin D2 is a potential down-stream signal molecule of the p210(BCR/ABL) tyrosine kinase in CML. The altered expression of cyclin D2 may contribute to the over proliferation of CML cells.</p>

Growth and gene expression of leukemia cell after treated with methylation inhibitor 5-aza-2'-deoxycytidine / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of demethylation therapy of leukemia by 5-aza-2'-deoxycytidine (5-aza-CdR).</p><p><b>METHODS</b>By using MTT test, NBT reduction reaction and DNA agarose gel electrophoresis, changes in proliferation, differentiation and apoptosis were observed in K562, HL-60 and fresh leukemia cells after treated with 5-aza-CdR. The mRNA expressions of DNMTs, p15, p53 and bcl-2 were measured by RT-PCR. The status of p15(INK4B) gene methylation was examined by methylation-specific PCR (MSP-PCR).</p><p><b>RESULTS</b>The growth inhibition of K562, HL-60 and fresh leukemia cells displayed a dose and time-dependent manner after treated by 5-aza-CdR. The differentiation-inducing ability on HL-60 cells was obvious at 0.5 micromol/L of 5-aza-CdR. The up-regulation of p15 mRNA and p53 mRNA expression and down-regulation of bcl-2 mRNA expression were obvious as compared with the control, but the DNMTs expression was not significantly different from the control. The methylation status of p15 gene in fresh leukemia cells decreased gradually with increasing concentration of 5-aza-CdR.</p><p><b>CONCLUSION</b>The proliferation of leukemia cells was obviously inhibited by 5-aza-CdR, its mechanism maybe related to the up-regulation of p15 and p53 genes and down-regulation of bcl-2 gene. The decrease of p15 gene methylation was associated with the competitive inhibition of 5-aza-CdR.</p>

Proliferation regulation effect of cyclin G1 antisense oligodeoxy-nucleotides with liposomal transfection on HL-60 cell / 中国实验血液学杂志

To investigate the effect of cyclin G1 antisense oligodeoxynucleotide (ASON) with liposomal transfection on mediating proliferation of HL-60 cell, the cyclin G1 ASON with liposomal transfection was used in vitro in co-culture with HL-60 cell, the protein and mRNA expression levels of cyclin G1 were measured by immunocytochemistry assay and RT-PCR. The cell apoptosis was detected by electron microscopy, in situ cell apoptosis detection kit (POD), DNA gel electrophoresis and flow cytometry (FCM). The results showed that in the cyclin G1 ASON group the protein and mRNA expression of cyclin G1 were significantly inhibited as compared with sense oligodeoxynucleotide (SON) group and blank group. When the ASON concentration increased, the proliferation ratio of HL-60 cell and CFU of HL-60 were also significantly inhibited. There was apoptosis of HL-60 cell. In conclusion, cyclin G1 ASON can specifically inhibit its protein and mRNA expression levels as well as the HL-60 cell proliferations and can accelerate the apoptosis of leukemia cells with concentration-dependent effect of ASON.

Effect of cyclin G1 antisense oligodeoxynucleotides (ASON) on the growth of HL-60 cells in nude mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the inhibition effect of cyclin G(1) antisense oligodeoxynucleotides (ASON) on the growth of HL-60 cells in nude mice.</p><p><b>METHODS</b>(1) Nude mice were divided into control group, sense oligodeoxynucleotides (SON) group and ASON group. After (60)Co radiation, with HL-60 cells SON group and ASON group were subcutaneously innoculated; (2) The weight and volume of tumors were continually measured; (3) The morphology of tumor cells was observed by microscope; (4) The protein and mRNA expression levels of cyclin G(1) were determined by flow cytometry (FCM) and reverse transcription polymerase chain reaction (RT-PCR); (5) The cell apoptosis was detected by electron microscopy and FCM.</p><p><b>RESULTS</b>(1) The inhibition rate of tumor in ASON group was 69.4%. In ASON group, the wight and volume of tumor were significantly lower than those in SON group and control group. (2) The HL-60 cells in ASON group showed morphologically smaller nuclei, less mitosis, less heteromorphosis and apoptosis.</p><p><b>CONCLUSION</b>The cyclin G(1) ASON can inhibit the growth of HL-60 cells in nude mice and induce apoptosis.</p>

The clinical significance of cyclin B1 expression in adult acute leukemia patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical significance of cyclin B1 expression in adult acute leukemia (AL) patients.</p><p><b>METHODS</b>The expression of cyclin B1 and p21 and their cell cycle distribution were measured by flow cytometry in 85 adult patients with de novo AL, 10 continuous complete remission (CCR) AL and 17 normal controls (NC). The mRNAs of cyclin B1, p21 cip1 and proliferation cell nuclear antigen (PCNA) in patients and NCs were measured with semi-quantity reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Cyclin B1 protein expression in de novo AL patients was significantly higher than that in NC (P < 0.001). It was higher in relapsed patients than in NC (P < 0.05) but was lower than in de novo AL (P < 0.01). There was no difference between the remission cases and NC (P = 0.21), and between CCR patients and NC (P > 0.05). The cyclin B1 overexpression ratio was higher than that of NC. A negative correlation between the expression levels of cyclin B1 and P21 was observed (r = -0.266, P < 0.05). The cyclin B1 protein expression level was positively correlated with its mRNA level. The expression of cyclin B1 was positively correlated with proliferation index (PI) levels, and with PCNA levels (rPI = 0.7314, rPCNA = 0.7152). Remission rate was higher in high cyclin B1 expression patients than in normal cyclin B1 expression patients (P < 0.01), so did the relapse rate (P < 0.01). Patients with higher cyclin B1 expression had higher survival rate.</p><p><b>CONCLUSION</b>Cyclin B1 was overexpressed and abnormally distributed in cell cycle phases in de novo AL patients. Overexpression of cyclin B1 might be a favorable prognostic factor for patients with AL.</p>

Effect of liposomal transfection of cyclin A antisense oligodeoxynucleotide (ASON) on HL-60 cell proliferation and apoptosis / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the effect of liposomal transfection of cyclin A antisense oligodeoxynucleotide (ASON) on HL-60 cell proliferation and apoptosis.</p><p><b>METHODS</b>By liposomal transfection, cyclin A ASON was co-cultured with HL-60 cells, the cell growth curve was determined by MTT assay and cell apoptosis electron-microscopy in situ cell apoptosis detection kit (POD), the protein and mRNA of cyclin A and bcl-2 were measured by FACS and RT-PCR, the role of cyclin A ASON in the development of leukemia was tested by the tumor formation in nude mice.</p><p><b>RESULTS</b>(1) In the cyclin A ASON liposomal transfection group (group A), the proliferation of HL-60 cell was significantly inhibited as compared to those in cyclin A ASON group (group B) (68.9% vs 24.8%) (P < 0.01). (2) The expressions of cyclin A and bcl-2 of group A were significantly lower than those in the control group (1.1% vs 38.8%, P < 0.01; 21.9% vs 65.0%, P < 0.01, respectively), and the DNA ladder and apoptosis body was displayed. (3) In group A, the rate of tumor formation in nude mice was lower, the time for tumor formation was longer and the volume of tumor was smaller than those in control group.</p><p><b>CONCLUSION</b>Liposomal transfection of cyclin A ASON can inhibit in vitro proliferation of leukemia cells and induce in vivo apoptosis of the tumor cell, which might provide a new target for gene therapy.</p>

Blastic natural killer cell leukemia--one case report and review of literature / 中华血液学杂志

<p><b>OBJECTIVE</b>To report a case of blastic natural killer cell leukemia with an aggressive clinical course.</p><p><b>METHODS</b>The characteristics of blastic NK cell leukemia and its treatment were discussed with review of literatures.</p><p><b>RESULTS</b>After combination chemotherapy and spinal cord segmental radiotherapy, the patient entered hematological remission, but the extramedullary lesion remained unchanged.</p><p><b>CONCLUSION</b>Blastic NK cell leukemia has an aggressive clinical course with poor response to treatment and unfavorable prognosis.</p>

Effects of FPIL6/IL2 on the differentiation and proliferation of lymphoid in the ML-IC assay / 中国医学科学院学报

<p><b>OBJECTIVE</b>To develop an in vitro assay that allows the culture and identification of a single human bone marrow progenitor closely related to hematopoietic stem cell, which is more primitive than LTC-IC, and to find an efficient culture conditions for NK-IC expansion.</p><p><b>METHODS</b>Fusion protein IL6/IL2 was reconstructed and expressed in E. coli DH5 alpha. ML-IC was determined by watching if the single cell can give rise to secondary progenitors with both LTC-IC and NK-IC characteristics. LTC-IC frequency was determined by the CFC clonogenic methylcellulose assay. NK-IC frequency was determined by phenotyping CD56 positive NK cells. The effect of FPIL6/IL2 on the expansion of NK-IC was examined by comparing the colony number of NK cells before and after the culture.</p><p><b>RESULTS</b>After the initial 4-week expansion culture, we showed that (25.75 +/- 5.68)% of freshly sorted Lin-/34+/DRdim cells were able to generate functional NK-IC in one or more secondary FPIL6/IL2 cultures, whereas (6.81 +/- 1.97)% in the control. A total of 102 NK-IC cells were present when were cultured for 6-7 weeks in FPIL6/IL2 expansion medium, which was much higher than the 33 NK-IC cells in the control.</p><p><b>CONCLUSION</b>ML-IC assay will prove useful to assess a very primitive hematopoietic cell with multilineage generative capacity. FPIL6/IL2 is capable of initiating and promoting NK-IC expansion greatly in ex vivo cultures in terms of net-conservation and net proliferation.</p>

Clinical Prognostic Value of Expression of DNA Methyltransferases Genes in Children with Acute Leukemia / 实用儿科临床杂志

Objective To investigate the relationship between the expression of DNA methyltransferases(DNMT)and clinical prognosis in children with acute leukemia(AL).Methods The mRNA expressions of DNMT1,DNMT3A,DNMT3B,p15,mdrl were measured in 56 AL children and 20 normal controls by semi-quantitative reverse transcriptionpolymerse chain reaction.Results In 56 cases of children with AL,the positive rate of DNMT1 was 73.2%(41/56);the positive rate of DNMT3A was 67.9%(38 /56);the positive rate of DNMT3B was 64.3%(36/56).Thirty-one cases showed positive expressions of the 3 DNMT simultaneously,4 cases with negative expressionss imultaneously,21 cases with at least 1 positive expression of the DNMT,positive rate of p15 was 19.6%(11/56);positive rate of mdrl was 28.6%(16/56),all 3 simultaneous expressions of the 3 DNMT in AL children were significantly higher than those in normal controls(P