c-myc、c-myb基因在白血病骨髓基质细胞中的表达及相关性研究

本研究检测分析c-myc、c-myb原癌基因在白血病细胞和骨髓基质细胞(BMSC)中的表达及其相关性。用逆转录聚合酶链反应(RT-PCR)技术定量分析原癌基因c-myc、c-myb的表达水平,通过流式细胞术分析白血病细胞和BMSC的膜表面抗原,同时采用染色体显带技术分析细胞核型。结果表明:①c-myc、c-myb基因在白血病细胞及其BMSC中均有一定的表达,尤其是在染色体异常的白血病细胞中高表达,其均值分别为1.15±0.38和1.03±0.48,与对照组比较均具有统计学差异(P<0.05);在染色体异常的BMSC中其均值分别为2.08±0.82(P<0.05)和1.46±0.29(P<0.05);②在白血病细胞和BMSC中,c-myc mRNA、c-myb mRNA的表达水平均与血小板计数具有相关性;③在不同预后组中,c-myc mRNA、c-myb mRNA表达呈线性相关;④在白血病细胞及急性组BMSC中原癌基因c-myc、c-myb的表达存在正相关;⑤白血病细胞中c-myc的表达量分别与BMSC中c-myc、c-myb的表达量有相关性。结论:在白血病中降低c-myc或c-myb原癌基因的表达水平可能成为治疗白血病的一种治疗方案。     

Peroxisome proliferator-activated receptor ligands affect growth-related gene expression in human leukemic cells

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors. Three subtypes of PPARs (alpha, beta, and gamma) have been identified in different tissues. PPAR alpha and PPAR gamma ligands inhibit cell proliferation and induce differentiation in several human cell models. We demonstrated that both PPAR alpha (clofibrate and ciprofibrate) and PPAR gamma ligands (troglitazone and 15 deoxy-prostaglandin J2, 15d-PGJ2) inhibited growth, induced the onset of monocytic-like differentiation, and increased the proportion of G0/G1 cells in the HL-60 leukemic cell line. Moreover, 3 days after the treatment with 2.5 microM 15d-PGJ2, an increase in sub-G0/G1 population occurred, compatible with an induction of programmed cell death. To clarify the mechanisms involved in HL-60 growth inhibition due to the effects of PPAR ligands, we investigated their action on the expression of some genes involved in the control of cell proliferation, differentiation, and cell cycle progression such as c-myc, c-myb, and cyclin D1 and D2. Clofibrate (50 microM), ciprofibrate (50 microM), and 15d-PGJ2 (2.5 microM) inhibited c-myb and cyclin D2 expression, whereas they did not affect c-myc and cyclin D1 expression. Only troglitazone (5 microM) decreased c-myc mRNA and protein levels, besides decreasing c-myb and cyclin D2. The down-regulations of c-myb and cyclin D2 expression represent the first evidence of the inhibitory effect exerted by PPAR ligands on these genes. Moreover, the inhibition of c-myc expression by troglitazone may depend on a PPAR-independent mechanism.     

血小板疾病中原癌基因c-myc,c-myb的研究进展

原癌基因c-myc和c-myb对造血系统有重要的调节作用.c-myc基因表达的变化与细胞增殖、分化状态有关,其表达产物在调节细胞生长、分化或恶性转化中发挥作用.c-myb参与转录,影响细胞增殖、分化、凋亡.近年来,随着分子生物学、分子免疫学、细胞生物学等相关技术的日趋成熟,原癌基因c-myc、c-myb在造血调控中的研究逐渐增多.研究发现,巨核-红系祖细胞的定向分化及血小板的异常与其表达水平有关.常见的血小板疾病见于血小板减少症、血小板增多症等,此类疾病的病因和发病机制尚未明T.本文就c-myc和c-myb基因的结构、功能及其在血小板疾病中的表达水平与意义作-综述.     

Targeted delivery of antisense oligonucleotides in cancer.

Formulations of antisense oligonucleotides (asODNs) against c-myb or c-myc protooncogenes have been prepared by a new technique that sequesters cationic lipid in the interior of a lipid particle. This technique results in high loading efficiency for the asODNs, small particle size and good stability. When targeted against melanoma cells or neuroblastoma cells via anti-GD(2) coupled at the particle surface, increased cell binding to the cells could be demonstrated. Targeted formulations showed greater inhibition of cell proliferation compared to non-targeted formulations or free drug. Inhibition of cell proliferation was demonstrated to be due to down-regulation of c-myb or c-myc protein expression. The formulations have long-circulation times in vivo, and evaluation for in vivo antitumor activity is currently underway.     

Regulation of T lymphocyte proliferation. Interleukin 2-mediated induction of c-myb gene expression is dependent on T lymphocyte activation state

We previously reported that with time, after antigenic stimulation of antigen-regulated murine T lymphocyte clones, total IL-2-R expression decayed 10-50-fold, commensurate with a decline in the ability of the cells to proliferate to IL-2. However, late after antigenic stimulation, when the cells were refractory to the IL-2-proliferative stimulus, high levels of high affinity IL-2-R remained. In this report we further explore the basis of unresponsiveness to IL-2 in the quiescent clones. We show that the proto-oncogene c-myc is induced in the late cell population by IL-2 to comparable levels observed early after antigen stimulation. IL-2-dependent c-myb induction, however, is seen only early after activation but not in the late-activated population. Analysis of the IL-2-dependent expression of c-myb mRNA with time after antigenic stimulation showed that steadystate c-myb expression declines dramatically with kinetics closely paralleling a decay in IL-2-dependent proliferative ability. In contrast, steadystate c-myc expression remains high throughout this period. Expression of c-myb is critical for proliferation of these cells since antisense oligodeoxy-nucleotide to c-myb can inhibit their IL-2-dependent proliferation. We present evidence for a pathway of c-myb induction via the TCR that is independent of the IL-2/IL-2-R interaction. In addition, the inhibition of IL-2-R-induced c-myb expression by 2-aminopurine and enhanced induction of c-myb via the TCR demonstrate that TCR activation and IL-2-R activation lead to induction of c-myb by different mechanisms.     

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or beta-TG peptides for 18 h and effect on Factor V steady-state mRNA levels was determined in 600 individual cells by in situ hybridization. beta-TG peptide had no effect on FV mRNA levels, whereas a approximately 60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to greater than or equal 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may function as a negative autocrine regulator of human megakaryocytopoiesis.     

Amplification of c-myc gene and overexpression of c-Myc protein in breast cancer and adjacent non-neoplastic tissue.

BACKGROUND: Deregulated c-Myc expression and alterations of c-myc oncogene have been reported to play an important role in breast cancer tumorigenesis. We examined the relationship between c-Myc protein level, amplification of c-myc oncogene and commonly used clinical and pathologic factors. METHODS: The studies were conducted on 94 ductal and lobular cancers. Amplification of c-Myc was assessed by the semiquantitative multiplex PCR assay. The amount of c-Myc protein was estimated by the densitometry analysis of Western blots. RESULTS: Amplification of c-Myc was found in 21% of examined cancers. There was no association of c-myc amplification with established risk factors. Overexpression of c-Myc protein without c-myc amplification was associated with negative status of axillary lymph node. The size of lobular carcinoma displaying overexpression of c-Myc and the normal copy number of c-myc gene was significantly smaller than the size of tumor with elevated c-Myc and amplification of c-myc gene (p < 0.01). Within tumors displaying overexpression of c-Myc protein and c-myc gene amplification the size of ductal carcinoma was smaller than the size of lobular carcinoma (p < 0.007). CONCLUSION: Data presented in this study suggest that alterations of c-myc gene and c-Myc protein level might be related to breast cancer progression. The prognostic utility of elevated level of c-Myc protein associated with normal status of c-myc gene for patients with lobular carcinoma requires further studies.     

c-myc oncogene expression and clinical outcome in carcinoma of the cervix

The expression of the c-myc oncogene was studied in paraffin-embedded specimens of cervical biopsies using a monoclonal antibody which binds to the 62,000 Dalton protein encoded by the c-myc gene. A range of cervical cancers from intraepithelial neoplasia to advanced grade IV tumours were studied together with normal cervical biopsies; c-myc status was correlated to clinical progress. There was no correlation seen between the clinical stage of the disease at presentation and c-myc expression. The 15 patients with c-myc negative cervical cancers were shown to have better disease free (mean--95.4 mos) and total survival (mean 118.0--mos) compared to the 16 patients that were c-myc positive 28.4 and 48.4 mos respectively). The pattern of recurrence differed between the two groups with c-myc positive tumours more likely to develop extra pelvic metastatic disease. The c-myc status of cervical cancer offers a prognostic indicator that could be useful in guiding treatment decisions.     

Expression of c-myc, epidermal growth factor receptor and c-erbB-2 in human endometrial carcinoma and cervical adenocarcinoma.

Thirty specimens of human endometrial carcinoma (n = 23) and cervical adenocarcinoma (n = 7) have been analyzed for c-myc, epidermal growth factor receptor (EGFR) and c-erbB-2 by immunohistochemistry. In endometrial carcinomas, expression of c-myc was observed in all cases, EGFR in 21 of 23 cases (91.3%) and c-erbB-2 in 7 of 23 cases (30.4%). In cervical adenocarcinomas, expression of c-myc was seen in 5 of 7 cases (71.6%), EGFR in all cases and c-erbB-2 in 2 of 7 cases (28.6%). c-myc immunoactivity was observed as nuclear or cytoplasmic stain or both, EGFR as membrane and cytoplasmic stain, c-erbB-2 as membrane stain. There was no relationship between expression of these three oncogenes and clinical prognostic factors in the present study.     

Proto-oncogene, proliferating cell nuclear antigen, perforin and growth factor gene expression in peripheral blood mononuclear cells from patients with IgA nephropathy]

IgA nephropathy, which is widely recognized as one of the most common primary glomerulonephritides, is characterized by the constant presence of IgA in the glomerular mesangium. We investigated proto-oncogenes, proliferating cell nuclear antigen (PCNA), perforin, platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) mRNA expression in peripheral blood mononuclear cells (PBMC) obtained from patients with IgA nephropathy, patients with other types of glomerulonephritis and healthy age-matched controls. The majority of patients with IgA nephropathy showed elevated c-myc, c-fos, c-jun, c-raf, PCNA, perforin, PDGF-B chain, and IGF-I and -II mRNA expression in PBMC, while no these mRNA expression was detected in PBMC obtained from patients with other types of glomerulonephritis or normal controls. A positive correlation was noted between mRNA levels and urinary protein excretion. mRNA expression also correlated with the histopathological findings in the renal tissue of patients with IgA nephropathy. These studies suggest that abnormal regulation of proto-oncogenes, PCNA, perforin, PDGF and IGF gene expression in PBMC may be associated with the progression of IgA nephropathy and may be useful as an indicator of disease activity.     

Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides.

Restenosis after angioplasty is due predominantly to accumulation of vascular smooth muscle cells (VSMCs). The resistance of restenosis to pharmacological treatment has prompted investigation of genes involved in VSMC proliferation. We have examined the effect on VSMC proliferation of blocking expression of the c-myc proto-oncogene with antisense oligodeoxynucleotides, both in vitro and in a rat carotid artery injury model of angioplasty restenosis. Antisense c-myc oligodeoxynucleotides reduced average cell levels of c-myc mRNA and protein by 50-55% and inhibited proliferation of VSMCs when mitogenically stimulated from quiescence or when proliferating logarithmically (IC50 = 10 micrograms/ml). Corresponding sense c-myc, two-base-pair mismatch antisense c-myc, antisense alpha-actin or glyceraldehyde phosphate dehydrogenase oligodeoxynucleotides did not suppress c-myc expression or inhibit VSMC proliferation. Antisense c-myc inhibition was relieved by overexpression of an exogenous c-myc gene. After balloon catheter injury, peak c-myc mRNA expression occurred at 2 h. Antisense c-myc applied in a pluronic gel to the arterial adventitia reduced peak c-myc expression by 75% and significantly reduced neointimal formation at 14 d, compared with sense c-myc and gel application alone. We conclude that c-myc expression is required for VSMC proliferation in vitro and in the vessel wall. C-myc is a therefore a potential target for adjunctive therapy to reduce angioplasty restenosis.     

c-Myc antisense limits rat liver regeneration and indicates role for c-Myc in regulating cytochrome P-450 3A activity

Expression of c-myc protein is associated with cell proliferation. The present study uses antisense oligomers to inhibit c-myc expression in the regenerating rat liver after 70% partial hepatectomy (PH). Antisense phosphorodiamidate morpholino oligomers (novel DNA analogs) were administered i.p. immediately after surgery to block expression of c-myc within the first 24 h after PH. A 20-mer PMO complimentary to the c-myc mRNA at the translation start site was an effective sequence (AVI-4126, 5'-ACGTTGAGGGGCATCGTCGC-3'). A single i.p. dose of 0.5 mg/kg AVI-4126 caused reduction of the regenerating liver c-myc protein in a sequence-specific and dose-dependent manner. Inhibition of c-myc expression resulted in reduction of proliferating cell nuclear antigen and arrested cells in the G(0)/G(1) phase of the cell cycle. The ratio of G(2):G(0) cell populations in the regenerating liver 24 h after PH dropped from 29.1 in saline vehicle-treated rats to 18.0 in rats treated with 2.5 mg/kg AVI-4126. The expression of cell cycle checkpoint protein p53 was inhibited with increasing doses of AVI-4126, but expression of p21(waf-1) was unaffected. The activity of cytochrome P-450 3A2 (CYP3A2) was evaluated by immunoblot analysis and erythromycin N-demethylation. AVI-4126 did not alter CYP3A activity in nonhepatectamized animals but showed a dose-dependent decrease in PH rats. We conclude that AVI-4126, antisense oligomer to c-myc, can reduce cell proliferation in the regenerating rat liver. Furthermore, inhibition of c-myc may indirectly influence the expression of CYP3A.     

Exportin 1 inhibition attenuates nuclear factor-kappaB-dependent gene expression

Activation of nuclear factor (NF)-kappaB is mediated by signal-induced phosphorylation of IkappaBalpha, subsequent IkappaBalpha degradation, and then translocation of unbound NF-kappaB to the nucleus. Termination of gene expression occurs when IkappaBalpha binds NF-kappaB subunits (Rel A) in the nucleus. Leptomycin B specifically inhibits export of IkappaBalpha and the inactive IkappaBalpha/Rel A complex via the nuclear export protein exportin 1. We hypothesized that inhibition of IkappaBalpha nuclear export would increase nuclear IkappaBalpha and attenuate NF-kappaB inflammatory gene expression in pulmonary microvascular endothelial cells. We found that inhibition of exportin 1 causes nuclear accumulation of both endogenous NF-kappaB (Rel A) and IkappaBalpha. IL-1beta causes nuclear accumulation of NF-kappaB (Rel A) but does not increase nuclear IkappaBalpha. Inhibition of exportin 1 before IL-1beta prevented an increase in the nuclear ratio of NF-kappaB (Rel A) to IkappaBalpha and decreases NF-kappaB DNA binding. Furthermore, inhibition of exportin 1 attenuates IL-1beta-induced phosphorylation of IkappaBalpha without affecting IkappaB kinase phosphorylation. Lastly, inhibition of exportin 1 attenuates monocyte chemoattractant protein, IL-8, and intercellular adhesion molecule expression in response to IL-1beta stimulation. We suggest that the decrease in cell activation due to exportin 1 inhibition is a result of termination of NF-kappaB DNA binding due to increased concentration of IkappaBalpha in the nucleus.     

c-myb反义寡脱氧核苷酸对白血病细胞HL-60的生物效应

为了研究c-myb在髓系白血病细胞的增殖和分化中所起的调控作用,合成了与c-myb mRNA翻译起始区域互补的一段反义寡脱氧核苷酸(18bp),将其作用于白血病细胞系HL-60细胞,观察到细胞生长抑制率平均为55%。经过c-myb反义寡脱氧核苷酸片段作用HL-60细胞,c-myb mRNA的表达不能被RT-PCR方法检测到,而在未加反义序列组和加随机序列组细胞中则能检测c-myb的表达。NBT染色后在Wright-Giemsa细胞涂片上可观察到未经处理和随机序列处理的95%HL-60细胞处于未分化阶段,经过c-myb反义寡脱氧核苷酸作用后的细胞中NBT阳性细胞占50％,部分细胞出现分化状态。