Elevated c-myc expression in progeria fibroblasts

Protooncogene expression was determined in cultured human fibroblasts derived from donors diagnosed with Hutchinson-Gilford Syndrome (Progeria). An increased level of c-myc expression was noted in progeria cells compared to normal parental controls and unrelated, unaffected, age-matched controls. In two progeria cultures a 150% increase in c-myc expression was observed, while a third progeria culture, subsequently identified as a non-classic progeria showed a 45% increase in c-myc expression. Southern blot analysis did not indicate an amplification or translocation of the c-myc gene. No significant differences were found in the expression of the c-erbB or c-src protooncogenes.     

人完全性葡萄胎中c-myc的表达及意义

目的研究c-myc基因在人完全性葡萄胎中的表达及其意义。方法取人完全性葡萄胎30例,正常早孕流产标本10例,用SABC免疫组织化学染色方法,检测c-myc基因在两种组织中的表达情况,并采用图像分析技术,对正常早孕绒毛组和完全性葡萄胎组c-myc的表达情况进行对比分析。结果与正常绒毛相比,c-myc基因在完全性葡萄胎组织中的表达量和表达的空间特异性有明显不同。结论 c-myc基因可能与完全性葡萄胎的发生密切相关。     

Preparation and characterization of specific antisera directed against different polypeptide domains encoded by the c-myc oncogene for studying the expression of this gene introduced into quail or rat cells

By using bacterial expression vectors, we have prepared antisera directed against two polypeptidic domains encoded by exons 2 and 3 of the human c-myc oncogene. These antisera which detect specifically the human c-myc proteins allow us to analyse the expression of human c-myc gene activated by retroviral sequences and introduced in quail embryo cells (QEC) or in established rat embryo fibroblastic cell line (208 F). Although human myc mRNA are expressed in the two cell types, the p64/p67 human c-myc proteins are only detected in the QEC.     

Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas.

We have determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myc genes contained missense mutations. This strongly supports the notion that the c-myc proto-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.     

c-myc oncogene expression inhibits the initiation of myogenic differentiation

The role of c-myc oncogene expression in myogenic differentiation has been established by transfecting rat myoblasts of the L6 cell line with plasmid pMT-myc, in which the c-myc coding sequences were under the control of the metallothionein I promoter. We observed that the constitutive expression of the exogenous c-myc gene inhibits muscular differentiation. A diminution of the endogenous c-myc gene expression occurs within the first 24 h after the transfer of the cells to a differentiating medium. This early decrease of c-myc expression is required for cell differentiation to occur. We have also observed that exogenous myc gene expression has no effect on endogenous myc expression.     

Silencing of human c-myc oncogene expression by poly-DNP-RNA

Deregulation of c-myc oncogene expression drives the progression of many different types of cancer. Recent experimental data suggest that even brief inhibition of c-myc expression may be sufficient to permanently stop tumor growth and induce regression of tumors. Previous efforts in developing an inhibitor to silence the c-myc gene were hampered by low efficacy and lack of sequence specificity. Here, we report the synthesis of an antisense RNA inhibitor based on a new 21-nt sequence on a poly- DNP-RNA platform that can specifically inhibit cancer cell growth by silencing c-myc gene expression. Both c-myc mRNA and protein levels were significantly decreased in MCF-7 cells following treatment with this antisense DNP-RNA inhibitor. The control compounds with sense or mismatched sequence were inactive. When c-myc transgenic mice were each treated with a single dose of the antisense RNA inhibitor, in vivo silencing of c-myc gene expression was observed for up to 72 hours by real-time RT-PCR. Similar treatment of c-myc transgenic mice with unmodified (native) homologous small interfering RNA (siRNA) had no effect on the mRNA concentration of the c-myc gene. Injection of this short antisense poly-DNP-RNA into mice did not induce the synthesis of DNP-binding immunoglobulins in the host. The observed in vivo gene silencing by this antisense RNA inhibitor suggests its possible use as a therapeutic agent for cancers involving the deregulation of c-myc gene expression.     

Relationship of c-myc gene copy number and gene expression: cellular effects of elevated c-myc protein

The relationship between gene copy number and expression and cellular consequences of elevated levels of c-myc protein has been investigated using recombinant Chinese hamster ovary cell lines transfected with DNA coding for the murine c-myc gene. HC-8 and LC-5 recombinant cells carry approximately 800 and 50 copies of c-myc sequences, respectively, under control of an inducible heat shock promoter. Multivariate flow cytometric analysis and clonogenic assays were used to measure the relationship among c-myc expression, rate of DNA synthesis, and cell survival. Following heat exposure, maximally induced HC-8 cells produced approximately tenfold more c-myc protein than heated LC-5 cells, suggesting a close relationship between gene copy number and level of expression. However, considerable heterogeneity in the level and time of c-myc expression was observed following heat induction, even though the amounts of genomic c-myc were relatively constant. Heterogeneity in gene expression was not attributable to variation in heat induction methodologies and/or cell cycle phase distributions. The presence of high levels of recombinant c-myc protein was associated with a decreased rate of bromodeoxyuridine incorporation into DNA. High levels of c-myc protein in HC-8 cells were inversely correlated with cell survival postheating, suggesting that high levels of c-myc protein are incompatible with cell survival.     

Erythroid differentiation and regulatory gene expression are modulated by adenosine derivatives interfering with S-adenosylmethionine metabolic pathway

The differentiation of murine erythroleukemia cells and the expression of SCL, Id1 and c-myc regulatory genes were studied. The first gene is a positive regulator of differentiation, while the other two are both negative regulators of differentiation and positive regulators of proliferation. Accordingly, our data show that when differentiation is stimulated SCL is upregulated while Id1 and c-myc are, coordinately, downregulated. The cultures were treated with two adenosine derivatives, 3-deazaadenosine and 3-deazaaristeromycin, known to act on the metabolic pathway of the methyl donor S-adenosylmethionin, in order to assess the possibility of a coordinated modulation, by these drugs, of regulatory gene expression and erythroid cell differentiation. 3-Deazaaristeromycin caused the simultaneous downregulation of Id1 and c-myc, whereas 3-deazaadenosine caused their upregulation; both drugs produced a transient increase in SCL expression. The use of these drugs evidenced a predominant regulatory effect of negative regulators in the control of erythroid differentiation. The distinct effects of the two drugs on regulatory gene expression led to an increased differentiation induced by 3-deazaaristeromycin and to a reduced differentiation induced by 3-deazaadenosine, if compared with controls. Southern analysis of DNA digested with methylation-specific restriction endonucleases showed that the administration of 3-deazaaristeromycin resulted in hypomethylation of SCL and c-myc, thus evidencing, in these cells, a clear correlation between DNA hypomethylation and differentiation but no straightforward correlation between DNA methylation and gene expression.     

Interleukin-6- and leukemia inhibitory factor-induced terminal differentiation of myeloid leukemia cells is blocked at an intermediate stage by constitutive c-myc.

Interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), two multifunctional cytokines, recently have been identified as physiological inducers of hematopoietic cell differentiation which also induce terminal differentiation and growth arrest of the myeloblastic leukemic M1 cell line. In this work, it is shown that c-myc exhibited a unique pattern of expression upon induction of M1 terminal differentiation by LIF or IL-6, with an early transient increase followed by a decrease to control levels by 12 h and no detectable c-myc mRNA by 1 day; in contrast, c-myb expression was rapidly suppressed, with no detectable c-myb mRNA by 12 h. Vectors containing the c-myc gene under control of the beta-actin gene promoter were transfected into M1 cells to obtain M1myc cell lines which constitutively synthesized c-myc. Deregulated and continued expression of c-myc blocked terminal differentiation induced by IL-6 or LIF at an intermediate stage in the progression from immature blasts to mature macrophages, precisely at the point in time when c-myc is normally suppressed, leading to intermediate-stage myeloid cells which continued to proliferate in the absence of c-myb expression.     

牛c-myc基因的克隆及其在皮肤成纤维细胞中的表达

为了构建包含牛c-myc基因编码序列的重组载体,以胎牛原始生殖嵴为材料,用RT-PCR方法克隆出牛c-myc 基因的编码序列,将其亚克隆至pMD19-T载体,再从酶切鉴定和测序正确的质粒上切下目的片段,定向克隆到pIRES2-AcGFP1-Nuc表达载体上,挑选序列正确的重组真核表达质粒转染牛皮肤成纤维细胞,用RT-PCR和Western blotting分别检测c-myc mRNA和蛋白的表达。结果表明,从胎牛原始生殖嵴中正确克隆了c-myc基因的全长编码序列,所构建的重组质粒能够在皮肤成纤维细胞中有效