Expression of c-fos and c-myc genes in uterus and nonreproductive organs during the implantation period in the rat.

The c-fos and c-myc mRNA levels were investigated in the uterus and nonreproductive organs of rats during the implantation period (from day 5 to 7 of pregnancy). They were determined by densitometric analysis of slot blots and the mean values (n = 4) at a defined age of pregnancy were compared with those observed in nonpregnant control rats (NP group). No significant variations of c-fos level were observed in the liver and brain of pregnant rats. In the uterus, the c-fos level decreased, reached a minimum on day 6 (4-fold decrease compared with the NP group), and then increased. The c-fos level significantly increased in the kidney (2- and 2.5-fold on days 6 and 7), and in the lung (2.5-fold on day 7). The c-myc level did not significantly vary in the uterus, liver, and lung. It significantly increased in the kidney (3-fold on day 7) and in the brain (4.1-, 3.8-, and 6-fold, on days 5, 6, and 7). The changes in c-fos and (or) c-myc gene expression in the uterus and nonreproductive organs could be due to sexual steroids and (or) systemic factors from uterine cells or blastocysts.     

Functional analysis of c-Myb protein in T-lymphocytic cell lines shows that it trans-activates the c-myc promoter.

The function of c-Myb protein was revealed by transfecting an expression vector containing the entire c-Myb protein-coding sequence into the murine CTLL-2 T-cell line. Expressions of high levels of c-Myb protein did not alter the expression of several T-cell markers, c-fos mRNA expression, responses to interleukin-2, and growth characteristics of these cells. Interestingly, expression of the c-myc gene was drastically increased in this clone. Further, the c-myb expression plasmid, but not a frameshift mutant of c-myb, enhanced the expression of a hybrid construct of c-myc promoter linked to a reporter gene by 8- to 14-fold. These results demonstrate a role of c-Myb protein in c-myc gene expression.     

PC-1在前列腺癌细胞中促进c-myc基因的表达

前列腺癌相关基因PC-1(Prostate and colon gene1)是属于癌基因D52家族成员,具有促进前列腺癌细胞雄激素非依赖性生长的功能。为了研究PC-1发挥这种生物功能的分子机制,文章在PC-1高表达的LNCaP-pc-1及对照LNCaP-zero细胞中,利用RT-PCR和Western blotting等方法检测c-myc基因表达;提取两细胞胞质和胞核蛋白,利用Western blotting分析c-myc上游调节蛋白β-catenin变化;利用c-Myc蛋白抑制剂10058-F4作用前列腺癌细胞C4-2,Western blotting检测PC-1蛋白表达变化。发现PC-1促进c-myc基因表达,并促进β-catenin入核;c-Myc蛋白抑制剂10058-F4可抑制PC-1的表达。结果表明:PC-1在前列腺癌中促进c-myc基因的表达,并且这种促进作用可能是通过Wnt/β-catenin信号通路实现的。同时,PC-1与c-Myc蛋白间可相互促进,进一步促进前列腺癌细胞雄激素非依赖性生长。     

Defective RNA-mediated c-myc gene silencing pathway in Burkitt's lymphoma

Keeping in view the fact that molecular basis of Burkitt's lymphoma (BL) is poorly understood, we attempted to explore the small interfering RNA (siRNA) mediated c-myc gene regulation using BL-derived EB-3 cell line as archetype cellular model. Such a study revealed that EB-3 cells possess 4-fold higher expression of Dicer gene coupled with 2-fold higher activity of RNA polymerase III than that observed in normal human lymphocytes. siRNAs derived from EB-3 cells had the inherent capacity to suppress c-myc gene expression in normal cells but not in native cells. Based on these findings we have proposed a novel RNA-mediated c-myc gene regulation pathway that may be responsible for BL.     

Down-regulation of c-myc and c-Ha-ras gene expression by tiazofurin in rat hepatoma cells

There was an overexpression of the c-myc gene (11-fold) and of the c-Ha-ras gene (2-fold) in rat hepatoma 3924A cells compared to normal rat liver as measured by dot-blot analysis of total cytoplasmic RNA. The overexpression of c-myc was attributed to a 10- to 14-fold amplification and rearrangement of the c-myc sequences as determined by Southern blot analysis. The expression of the c-myc also was dependent upon the proliferative state of the hepatoma cells. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide; NSC 286193), an inhibitor of the activity of IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of GTP biosynthesis, resulted in a rapid drop (less than 1 h) to 50% of control in the target enzyme activity in the hepatoma cells and in a subsequent marked decrease to 55% in GTP concentration. These events were followed at 12 h of tiazofurin treatment by a 3-fold reduction in the expression of the c-myc gene and a 9-fold decline in that of the c-Ha-ras gene. These results in the hepatoma cells provide evidence in support of the earlier demonstrated correlation in K562 cells between GTP concentration and expression of c-myc and c-ras genes (Olah et al., 1989). These genes might depend on GTP for their expression in hepatoma cells and they might cooperate in a signal pathway that controls cell proliferation.     

Ku80 binds to human replication origins prior to the assembly of the ORC complex

The Ku heterodimer, an abundant nuclear protein, binds DNA replication origins in a sequence-specific manner and promotes initiation. In this study, using HCT116 Ku80+/- haplo-insufficient and Orc2(delta/-) hypomorphic cells, the order of binding of Ku and the human origin recognition complex (HsORC) was determined. The nuclear expression of Ku80 was found to be decreased by 60% in Ku80+/- cells, while its general association with chromatin was decreased by 33%. Coimmunoprecipitation studies indicated that the Ku heterodimer associates specifically with the human HsOrc-2, -3, -4, and -6 subunits. Chromatin immunoprecipitation (ChIP) experiments, using cells synchronized to late G1, showed that the association of Ku80 with the lamin B2, beta-globin, and c-myc origins in vivo was decreased by 1.5-, 2.3-, and 2.5-fold, respectively, in Ku80+/- cells. The association of HsOrc-3, -4, and -6 was consistently decreased in all three origins examined in Ku80+/- cells, while that of HsOrc-2 showed no significant variation, indicating that the HsOrc-3, -4, and -6 subunits bind to the origins after Ku80. In Orc2(delta/-) cells, the association of HsOrc-2 with the lamin B2, beta-globin, and c-myc origins was decreased by 2.8-, 4.9-, and 2.8-fold, respectively, relative to wild-type HCT116 cells. Furthermore, nascent strand abundance at these three origins was decreased by 4.5-, 2.3-, and 2.6-fold in Orc2(delta/-) relative to HCT116 cells, respectively. Interestingly, the association of Ku80 with these origins was not affected in this hypomorphic cell line, indicating that Ku and HsOrc-2 bind to origins independently of each other.     

Hormonal regulation of thyroid peroxidase in normal and transformed rat thyroid cells

The hormonal induction of thyroid peroxidase (TPO) mRNA is studied in the functional rat thyroid cell line FRTL-5 and compared to the induction of thyroglobulin (TG) mRNA and I- uptake. TPO and TG mRNAs are regulated by TSH and by insulin-like growth factor I (IGF-I) and/or insulin. However, while TPO is more sensitive to TSH regulation (5- to 6-fold increase vs. 2- to 3-fold increase by IGF-I), TSH and IGF-I are equally potent in increasing TG mRNA levels (3- to 4-fold). Regulation of I- uptake appears to be different: thus TSH greatly (15-fold) increases I- uptake, while IGF-I or insulin are completely ineffective. TPO and TG mRNAs and I- transport display different sensitivity to transformation of rat thyroid cells. Thus, when another differentiated rat thyroid cell line, the PC cells, are transformed by human c-myc (PC myc), TPO and TG mRNAs are both present at normal levels, while I- uptake is slightly decreased; in the PC cells transformed by polyomavirus middle-T-antigen (PC PyMLV) TPO mRNA is undetectable and I- uptake is greatly decreased, while TG mRNA is present at normal levels. All three differentiated functions are switched off in PC cells transformed by the cooperation of c-myc and polyomavirus middle-T-antigen (PC myc + PyMLV).     

UV damage and repair in the domain of the human c-myc oncogene

We have used a modification of the Southern hybridization method to analyze the removal of UV-induced pyrimidine cyclobutane dimers from the domain of the c-myc oncogene. The study was performed in human COLO320HSR cells, which exhibit a 30- to 40-fold amplification of c-myc that is maintained in a marker chromosome as a homogeneously staining region. Intron 2 and the region upstream from the gene showed better dimer removal than intron 1 or the region downstream from the c-myc gene. Regions showing less repair coincide with regions that are hotspots for mutations and chromosome translocations. Therefore, it is proposed that the inefficiency of DNA repair may play an important role in the origin of c-myc rearrangements.     

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.     

受四环素调控的真核表达系统SK-Hep1细胞株的建立

建立稳定、高效表达外源基因的SK-Hep1细胞株,以便进一步研究基因的作用.首先将调控质粒pCDNA6/TR转染SK-Hep1细胞,经潮霉素筛选得到多个稳定单克隆.各个单克隆分别扩大培养后,转染pCDNA4/TO/lacZ质粒,再经过DOX(强力霉素)诱导表达,检测β-半乳糖苷酶(β-D galaetosidase,β-gal)活性,从而筛选出高诱导水平低背景表达的SK-Hep1 tet-on细胞株.最后,再将pCDNA4/TO/c-myc质粒转染进SK-Hep1 tet-on细胞株,进一步通过Western blotting检测该系统对下游基因的表达调控.成功建立了一株受DOX调控的高诱导水平低背景表达的细胞株SK-Hep1 tet-on 10#.