增殖细胞核抗原（PCNA）与白血病和MDS相关性的研究

我们采用ＰＣＮＡ基因探针对人白血病细胞株ＨＬ－６０和Ｕ９３７、急性白血病、骨髓增生异常综合征和贫血患者骨髓细胞中ＰＣＮＡ ＲＮＡ的含量进行了研究，实验结果表明，ＰＣＮＡ基因的表达水平在急性白血病最高，ＭＤＳ－ＲＡ介于白血病和贫血之间。提示ＰＣＮＡ基因的表达水平与细胞的增殖及潜在恶性变程度有关，此外，我们还注意到，ＰＣＮＡ的表达水平与急性白血病患者的性别、年龄、达到完全缓解所需的时间及复发等因素无明     

Knockdown of IARS2 Inhibited Proliferation of Acute Myeloid Leukemia Cells by Regulating p53/p21/PCNA/eIF4E Pathway

IARS2 encodes mitochondrial isoleucine-tRNA synthetase, which mutation may cause multiple diseases. However, the biological function of IARS2 on acute myeloid leukemia (AML) has not yet been identified. In the present study, qRT-PCR was used to determine the expression of IARS2 in K562, THP1, and HL-60 leukemia cells. Additionally the mRNA levels of IARS2 in CD34 cells and AML cells obtained from patients were detected by qRT-PCR. IARS2-shRNA lentiviral vector was established and used to infect acute myeloid leukemia HL-60 cells. qRT-PCR and Western blot analysis were employed to assess the knockdown effect of IARS2. The proliferation rate and cell cycle phase of HL-60 cells after IARS2 knockdown were evaluated by CCK-8 assay and flow cytometry. The PathScan Antibody Array was used to determine the expression of cell cycle-related proteins in HL-60 cells after IARS2 knockdown. The expression of proliferation-related proteins in HL-60 cells after IARS2 knockdown was determined by Western blot analysis. Results showed that IARS2 expression was stable and much higher in HL-60, THP-1, and K562 leukemia cells and AML cells obtained from patients than that of human CD34 cells. Compared with cells of the shCtrl group, IARS2 was markedly knocked down in cells that were transfected with lentivirus encoding shRNA of IARS2 in HL-60 cells (p<0.05). IARS2 knockdown significantly inhibited the proliferation and induced cycle arrest at the G₁ phase in HL-60 cells. Additionally IARS2 knockdown significantly increased the expression of p53 and p21, and decreased the expression of PCNA and eIF4E in HL-60 cells. In conclusion, IARS2 knockdown can inhibit acute myeloid leukemia HL-60 cell proliferation and cause cell cycle arrest at the G₁ phase by regulating the p53/p21/PCNA/eIF4E pathways.     

塞来昔布对NB4细胞增殖、凋亡及VEGF表达的影响

目的 探讨塞来昔布对急性早幼粒细胞白血病细胞的抑制作用及可能机制.方法 不同浓度塞来昔布处理急性早幼粒细胞白血病细胞株NB4,MTT法检测塞来昔布作用后细胞的增殖情况.采用流式细胞术测定NB4细胞周期分布及凋亡;Western blot检测细胞凋亡相关蛋白Bcl-2表达以及RTPCR检测塞来昔布作用对VEGF、HIF-1α在mRNA水平的表达.结果 MTT显示塞来昔布对NB4细胞生长有明显抑制作用.20～80 μmol/L塞来昔布作用24 h,NB4细胞G0/G1期比例明显升高,而S期细胞比例下降(P＜0.05);塞来昔布处理组细胞凋亡率明显高于对照组,Western blot显示Bcl-2蛋白表达下调(P＜0.05);RT-PCR显示塞来昔布可抑制VEGF及HIF 1α的mRNA表达.结论 塞来昔布在体外可抑制急性早幼粒细胞白血病细胞增殖并诱导凋亡.     

Leukemic stromal hematopoietic microenvironment negatively regulates the normal hematopoiesis in mouse model of leukemia

Background and Objective: Leukemic microenvironment has a major role in the progression of leukemia. Leukemic cells can induce reversible changes in microenvironmental components, especially the stromal function which results in improved growth conditions for maintaining the malignant leukemic cells. This study aimed to investigate the survival advantage of leukemic cells over normal hematopoietic cells in stromal microenvironment in long term. Methods: The mice were injected intraperitoneally with N-N’ eth...     

Leptin Receptor and Leukemia

The receptor for leptin, the gene product of the obese gene, is expressed in hematopoietic stem cells. Leptin stimulates normal myeloid and erythroid development, and is secreted from bone marrow adipocytes, which occupy most of the marrow cavity in humans. Leptin might thus play an important role in the control of the expansion and differentiation of primitive hematopoietic cells through paracrine interaction in the bone marrow microenvironment. Leukemic cells of some patients with acute myeloblastic leukemia, acute lymphoblastic leukemia, and chronic myeloid leukemia (CML) also express the leptin receptor. In cases of CML, higher expression of leptin receptor is observed during blast crisis than in chronic phase. Leptin alone and in combination with other cytokines has stimulative effects on proliferation of leukemia cells as well as anti-apoptotic effects. These findings suggest the possibility that leptin plays roles in the pathophysiology of leukemia.     

Expression of the Annexin VIII Gene Acute Promyelocytic Leukemia

Annexin VIII is preferentially expressed in APL, but its level of expression in other subtypes of AML is much lower. Annexin VIII was originally found to be a vascular anticoagulant, but evidence obtained from our recent studies suggests that it does not play a role in hemorrhage diathesis in APL. The specific expression of annexin VIII in APL may relate to its possible role in hematopoietic cell differentiation. The expression of annexin VIII is developmentally regulated in APL-derived NB4 cells. It can be downregulated as a response to induction by ATRA, an agent which is also capable of inducing maturation of NB₄ cells. Our current understanding is that annexin VIII is most likely involved in signal transduction and may have a role as a modulator of PKC. A change in cellular PKC activity is expected to have a significant impact on cell differentiation and proliferation. The biological function of annexin VIII is currently unknown, but its expression in APL and its possible role in differentiation and proliferation of the leukemia cells would provide an excellent model system to study and elucidate this intriguing question.     

Enhancement of the Cytotoxicity of Cytosine Arabinoside by Interleukin-3

We have evaluated the feasibility of enhancing the cytotoxic effect of cytosine arabinoside (ara-C) on acute myeloid leukemia (AML) cells by increasing the proliferative activity with hematopoietic growth factors. Leukemic cells from 8 persons with AML were tested. Preincubation with interleukin (IL)-3 (5 U/ml) for 3 days increased DNA synthesis as measured by tritiated thymidine incorporation and Ki67 expression in cells from 7 out of 8 persons with AML, Leukemic cells preincubated with IL-1 (10 U/ml) or IL-3 (5 U/ml) were subsequently exposed to ara-C (3μg/ml) for the final 24 h and the activity of ara-C against clonogenic acute myeloid leukemia cells was evaluated in terms of the inhibition of colony formation in semisolid media. The exposure to ara-C inhibits the proliferation of a higher proportion of clonogenic cells in culture pretreated with IL-3 than in control or cells pretreated with IL-1. The enhanced cytotoxic effect of ara-C in the cells pretreated with IL-3 correlated with increased formation of intracellular ara-CTP. IL-3-induced recruitment of quiescent blasts into the proliferative compartment will lead to increased formation of ara-CTP in the cells, which would result in an enhanced leukemia cell kill.     

Levels of p53 protein increase with maturation in human hematopoietic cells

Transfection of the wild-type p53 gene into malignant cell lines usually results in an inhibition of proliferation. However, the physiological function of the endogenous p53 gene product has been difficult to ascertain. In order to examine whether p53 is involved in the regulation of proliferation and/or differentiation of hematopoietic tissue, we modified a recently developed flow cytometric assay to assess p53 protein expression in normal human hematopoietic cells, primary leukemias, and selected leukemia cell lines. In normal human bone marrow, p53 protein was not detected in the proliferative, progenitor cell populations identified by the cell surface antigens CD34 (progenitor cells of multiple lineages) or glycophorin (erythroid precursors). In contrast, low but detectable levels of p53 protein were observed in the nonproliferative, mature lymphoid, granulocytic, and monocytic cell populations. Similarly, p53 levels increased and DNA synthesis decreased during 12-O-tetradecanoylphorbol-13-acetate-induced differentiation of ML-1 myeloblastic leukemia cells. Both of these results suggest that endogenous, wild-type p53 protein may play a role in hematopoietic cell maturation, possibly by contributing to the inhibition of proliferation that occurs during terminal differentiation. Leukemia cells deviated from this pattern of expression: (a) in contrast to the normal, proliferative bone marrow progenitor cells, a significant percentage of patient leukemia samples expressed detectable levels of p53 protein; and (b) leukemia cell lines exhibited lineage-specific abnormalities in p53 expression, with overexpression in lymphoid cell lines and lack of expression in myeloid cell lines.     

CXXC5 (Retinoid-Inducible Nuclear Factor,RINF) is a Potential Therapeutic Target in High-Risk Human Acute Myeloid Leukemia

The retinoid-responsive gene CXXC5 localizes to the 5q31.2 chromosomal region and encodes a retinoid-inducible nuclear factor (RINF) that seems important during normal myelopoiesis. We investigated CXXC5/RINF expression in primary human acute myeloid leukemia (AML) cells derived from 594 patients, and a wide variation in CXXC5/RINF mRNA levels was observed both in the immature leukemic myeloblasts and in immature acute lymphoblastic leukemia cells. Furthermore, patients with low-risk cytogenetic abnormalities showed significantly lower levels compared to patients with high-risk abnormalities, and high RINF/CXXC5/ mRNA levels were associated with decreased overall survival for patients receiving intensive chemotherapy for newly diagnosed AML. This association with prognosis was seen both when investigating (i) an unselected patient population as well as for patients with (ii) normal cytogenetic and (iii) core-binding factor AML. CXXC5/RINF knockdown in AML cell lines caused increased susceptibility to chemotherapy-induced apoptosis, and regulation of apoptosis also seemed to differ between primary human AML cells with high and low RINF expression. The association with adverse prognosis together with the antiapoptotic effect of CXXC5/RINF suggests that targeting of CXXC5/RINF should be considered as a possible therapeutic strategy, especially in high-risk patients who show increased expression in AML cells compared with normal hematopoietic cells.     

PDCD2 functions in cancer cell proliferation and predicts relapsed leukemia

PDCD2 is an evolutionarily conserved eukaryotic protein with unknown function. The Drosophila PDCD2 ortholog Zfrp8 has an essential function in fly hematopoiesis. Zfrp8 mutants exhibit marked lymph gland hyperplasia that results from increased proliferation of partially differentiated hemocytes, suggesting Zfrp8 may participate in cell growth. Based on the above observations we have focused on the role of PDCD2 in human cancer cell proliferation and hypothesized that aberrant PDCD2 expression may be characteristic of human malignancies. We report that PDCD2 is highly expressed in human acute leukemia cells as well as in normal hematopoietic progenitors. PDCD2 knockdown in cancer cells impairs their proliferation, but not viability relative to parental cells, supporting the notion that PDCD2 overexpression facilitates cancer cell growth. Prospective analysis of PDCD2 in acute leukemia patients indicates PDCD2 RNA expression correlates with disease status and is a significant predictor of clinical relapse. PDCD2’s role in cell proliferation and its high expression in human malignancies make it an attractive, novel potential molecular target for new anti-cancer therapies.     

A combination of paclitaxel and siRNA-mediated silencing of Stathmin inhibits growth and promotes apoptosis of nasopharyngeal carcinoma cells

Background

Stathmin, a microtubule associated protein (MAP), is an important molecular target for cancer therapy. Paclitaxel is one of the primary antitumor drugs targeting microtubules (MTs). We hypothesized that decreasing the expression level of Stathmin might improve the effectiveness of paclitaxel in the treatment of nasopharyngeal carcinoma (NPC).

Methods

NPC cell lines, CNE1-LMP1 and HNE2, and a CNE1-LMP1 tumor xenograft mouse model were used to test both in vitro and in vivo our siRNA-based Stathmin silencing strategy. The effects of Stathmin silencing on cell proliferation, apoptosis, and viability were investigated using MTT, AO/EB staining, TUNEL, caspase protein detection, and FCM assays. Cell migration and invasion were assayed using a Transwell assay. The combined effects of Stathmin silencing and paclitaxel were investigated using MTT, FCM, Western blot and indirect immunofluorescence assays. The effect of paclitaxel on Stathmin expression in NPC cells and, in addition, A375, MGC and HeLa cells was determined by RT-PCR and Western blotting.

Results

We found that siRNA-mediated silencing of Stathmin suppresses proliferation, induces apoptosis through the mitochondrial pathway, and causes G2/M-phase cell cycle arrest in the NPC cell lines CNE1-LMP1 and HNE2. Also, the migration and invasion of the respective NPC cells were found to be inhibited. In addition, we show that a combination of Stathmin silencing and paclitaxel is more effective than either agent alone in inhibiting proliferation and inducing apoptosis, cell cycle arrest, and MT polymerization. Furthermore, we found that Stathmin expression in the tumor cells is down-regulated by paclitaxel treatment.

Conclusion

siRNA-mediated silencing of Stathmin suppresses the proliferation, invasion and metastasis, and induces the apoptosis of NPC cells. Paclitaxel reduces the expression of Stathmin, and combining Stathmin silencing with paclitaxel treatment enhances MT polymerization. This combined strategy may provide a new approach for clinical NPC treatment.     

布罗莫结构域4抑制剂JQ1对Ph阳性急性淋巴细胞白血病细胞增殖的抑制作用和机制研究

目的 观察布罗莫结构域4(brd4)抑制剂JQ1对Ph阳性急性淋巴细胞白血病(Ph+ALL)细胞株SUP-B15的增殖抑制、凋亡作用及对brd4及其下游基因myc、p53表达的影响,探讨其是否对bcr-abl有逆转作用.方法 采用不同浓度的JQ1处理SUP-B15细胞.四甲基偶氮唑蓝(MTT)法检测细胞的增殖抑制率;AnnexinV-FITC/PI荧光染色流式细胞术检测细胞凋亡率;实时荧光定量PCR(RT-PCR)法检测bcr-abl、brd4 、myc 、p53的mRNA表达.结果 不同浓度的JQ1均能抑制SUP-B15细胞的增殖,诱导细胞发生凋亡,凋亡率较对照组明显增高,并呈时间-剂量依赖性,作用72 h半数抑制浓度为1.0 μmol/L.同时JQ1可以使bcr-abl、 brd4、 myc的mRNA水平下降,而使p53 mRNA的水平升高.结论 brd4抑制剂JQ1可通过下调brd4的表达,影响其下游基因myc及p53的表达,同时逆转bcr-abl表达,达到抑制Ph+ ALL细胞增殖、诱导细胞凋亡的作用.     

Twist 1通过MPL促进髓系白血病细胞增殖和耐药

目的:检测Twist-1与骨髓增生性白血病病毒癌基因(myeloproliferative leukemia virus oncogene,MPL)在髓系白血病患者和髓系造血系统恶性肿瘤细胞系中表达的相关性,并探讨Twist-1是否通过MPL对髓系白血病细胞增殖、耐药发挥促进作用.方法:选取中国医学科学院血液病医院2005年1月至2008年12月初次诊断为急性髓系白血病(acute myeloid leu-kemia,AML)、慢性粒细胞白血病(chronic myeloid leukemia,CML)患者的骨髓标本41例(其中AML 23例、CML 18例),用Re-al-time PCR检测AML、CML患者以及髓系造血系统恶性肿瘤细胞系中Twist-1和MPL mRNA的表达情况,并分析其相关性.构建MPL过表达载体,制备慢病毒并感染髓系白血病细胞系K562、U937,通过细胞计数实验、集落形成实验以及药物敏感实验评价其对白血病细胞增殖、集落形成能力以及耐药的影响,并进一步确定Twist-1是否通过MPL发挥白血病促进作用.结果:在U937和K562细胞中过表达Twist-1明显增加MPL蛋白水平(P＜0.05),敲降Twist-1后MPL mRNA的表达水平明显下降(P＜0.01);AML、CML患者骨髓单核细胞(bone marrow mononuclear cells,BMMCs)中Twist-1 mRNA表达与MPL mRNA表达水平呈显著正相关(P＜0.05).过表达MPL使K562和U937细胞对化疗药物柔红霉素及伊马替尼的敏感性显著降低(P＜0.01),且提高K562-MPL、U937-MPL的细胞增殖、集落形成数目(均P＜0.01);干扰Twist-1并过表达MPL显著降低髓系白血病细胞增殖和集落形成(均P＜0.01).结论:Twist-1通过MPL促进AML、CML白血病细胞的增殖、存活和耐药.     

Inhibition of PRAME expression causes cell cycle arrest and apoptosis in leukemic cells

The preferentially expressed antigen of melanoma (PRAME) is known as a tumor-associated antigen, but its function in leukemia remains unclear. We investigated the function with small interfering RNA (siRNA)-induced knockdown of PRAME in a K562 cell line. After PRAME siRNA transfection, proliferation was suppressed and cell cycle analysis showed G₀/G₁ arrest, followed by apoptosis. PRAME siRNA-treated cells also showed changes in the genes affecting erythroid differentiation. We examined the PRAME expression levels and the S phase population of 32 acute leukemia patients at the time of diagnosis and relapse. An increase of the S phase population was accompanied by an increase of PRAME expression at relapse. Our results suggest that PRAME plays an important role in disease progression in acute leukemia.     

De Novo Acute Leukemia with a Sole 5q-: Morphological, Immunological, and Clinical Correlations

The 5 q deletion is frequently found in myelodysplastic syndromes and acute non lymphoid leukemia, but this anomaly is usually found in secondary diseases and associated with many other chromosomal aberrations. This report describes four cases of “de novo” acute leukemia with a sole 5q- anomaly. They had no cytological, genetic or clinical characteristics of secondary disorders. It is important to note that of the four patients studied, three had proliferation of immature blast cells. One case was classified as a MO AML and two as “undifferentiated” acute leukemia. Furthermore, these four cases of acute leukemia showed a deletion of the same portion of the long arm of chromosome 5: q22q33. On the same part of this chromosome many hematopoietic growth factor genes have been located, like IL3 and GM-CSF which have early undifferentiated hematopoietic stem cells as a their target.