Interleukin-1-inducible tumor growth arrest is characterized by activation of cell type-specific "early" gene expression programs.

Human melanoma cells, A375-C6, were "committed" to growth arrest within a few hours of exposure to interleukin-1 (IL-1). Co-treatment with actinomycin D rescued the cells from the "commitment," suggesting that "early" gene activation events may be crucial for growth arrest. To understand the mechanism of IL-1 action, we are studying early genes whose expression is induced by the cytokine. Five early genes associated with IL-1 action in the melanoma cells were isolated by differential screening of a cDNA library, which was enriched for sequences representing IL-1 responsive genes (IRGs). Nucleotide sequencing identified four of the genes as gro-alpha, gro-beta, c-jun and nur77/NGF1-B/NAK1, respectively, while the fifth was judged as novel by GenBank search and designated IRG-9. None of the early genes was uniquely associated with the antiproliferative action of IL-1: other growth-inhibitory as well as growth-stimulatory signals induced these genes in diverse cell types. However, analysis of the induction patterns of the IRGs and other well known early genes revealed that IL-1 action in the melanoma cells is characterized by activation of a unique primary gene expression program. This program was defined by the magnitude and temporal pattern of induction of the five IRGs, feeble induction of c-fos, and lack of induction of Egr-1 and c-myc. We present evidence that this program is growth arrest-specific in the melanoma cells and that distinct cell type-specific programs are associated with IL-1 growth-regulatory actions in other tumor cells. Based on these data, we propose that early genes may play multifunctional roles in tumor growth control, but specificity for the growth arrest action of IL-1 is determined by the composite early gene induction program.     

Induction of early response genes in trypsin inhibitor-induced pancreatic growth

Endogenous CCK release induced by a synthetic trypsin inhibitor, camostat, stimulates pancreatic growth; however, the mechanisms mediating this growth are not well established. Early response genes often couple short-term signals with long-term responses. To study their participation in the pancreatic growth response, mice were fasted for 18 h and refed chow containing 0.1% camostat for 1-24 h. Expression of 18 early response genes were evaluated by quantitative PCR; mRNA for 17 of the 18 increased at 1, 2, 4, or 8 h. Protein expression for c-jun, c-fos, ATF-3, Egr-1, and JunB peaked at 2 h. Nuclear localization was confirmed by immunohistochemistry of c-fos, c-jun, and Egr-1. Refeeding regular chow induced only a small increase of c-jun and none in c-fos expression. JNKs and ERKs were activated 1 h after camostat feeding as was the phosphorylation of c-jun and ATF-2. AP-1 DNA binding evaluated by EMSA showed a significant increase 1-2 h after camostat feeding with participation of c-jun, c-fos, ATF-2, ATF-3, and JunB shown by supershift. The CCK antagonist IQM-95,333 blocked camostat feeding-induced c-jun and c-fos expression by 67 and 84%, respectively, and AP-1 DNA binding was also inhibited. In CCK-deficient mice, the maximal response of c-jun induction and AP-1 DNA binding were reduced by 64 and 70%, respectively. These results indicate that camostat feeding induces a spectrum of early response gene expression and AP-1 DNA binding and that these effects are mainly CCK dependent.     

Mitogenic growth factors regulate differentially early gene mRNA expression: a study on two clones of 3T3 fibroblasts.

The relationship between cell proliferation and mRNA levels of the immediate early genes c-fos, c-jun, and jun B has been investigated in two clones of 3T3 fibroblasts (D1-3T3 and N2-3T3) upon treatment with basic fibroblast growth factor (bFGF), thrombin, phorbol 12-myristate 13-acetate (PMA) and dibutyryl cyclic AMP (Bt2cAMP). The 3T3-derived clone D1-3T3 almost stops dividing upon serum deprivation, while the N2-3T3 clone does not. The proliferation of the two clones was stimulated by thrombin and PMA and inhibited by Bt2cAMP. Basic FGF stimulated the growth of D1-3T3 but partly inhibited that of N2-3T3 cells. In spite of variable mitogenic response, immediate early genes, c-fos, c-jun, jun B, and c-myc, were induced by the growth factors and by PMA in both cell clones. In our experimental conditions the early gene mRNAs were expressed independently; i.e., the expression of one protooncogene had no bearing on the expression of the other. The cell growth was not directly related to the expression of a particular protooncogene mRNA. Data are presented showing that early gene mRNA expression induced by bFGF or thrombin was not mediated by protein kinase C activation while thrombin-induced mitosis was. Basic FGF induced a part of c-jun mRNA expression, but not mitosis, through a pertussis toxin-sensitive mechanism.     

FOS／JUN介导佛波酯对内皮素基因表达的诱导作用

利用凝胶电泳迁移率改变实验、RNA印迹和蛋白质印迹分析分别检查了c-jun抗体对内皮素-1(ET-1)基因AP-1位点与核蛋白结合的影响及肿瘤促进剂佛波酯(TPA)对c-fos/c-jun基因表达的作用.结果发现,c-jun抗体可使AP-1位点-核蛋白复合物的电泳迁移率发生改变,TPA显著促进c-fos/c-jun基因表达和血管内皮细胞的AP-1结合活性.实验表明,TPA对ET-1基因表达的诱导作用是通过促进AP-1转录因子c-fos/c-jun合成来介导的.     

Contribution of IL-6 to the antiproliferative effect of IL-1 and tumor necrosis factor on tumor cell lines

The role of IL-6 in the antiproliferative effect of IL-1 for tumor cell lines was investigated using IL-1-sensitive cell lines. Human recombinant IL-1 alpha and IL-6 both inhibited the growth of an IL-1-sensitive cloned human melanoma cell line (A375-C6). However, IL-1 has greater maximum growth inhibitory activity than IL-6. Conditioned medium of the tumor cells that were treated with IL-1 contained IL-6 as determined by ELISA. Northern blot analysis revealed that IL-6 mRNA expression increased in IL-1-treated cells. In addition, antibody against human IL-6 neutralized about 50% of the antiproliferative effect of IL-1. The growth of an IL-1-resistant clone of A375 cells (A375-C5), which cannot be shown to express any detectable IL-1R, was inhibited by IL-6 to the same degree as A375-C6 cells. The A375-C5 cell line did not produce IL-6 or increase IL-6 mRNA after stimulation with IL-1. These results indicate that IL-6 mediates in part the antiproliferative effect of IL-1 on A375-C6 cells by acting as an autocrine antiproliferative factor. IL-1 also inhibited the growth of a malignant human mammary cell line (MDA-MB-415). IL-6 exhibited only slight growth inhibition in this cell line. Neither IL-6 production nor IL-6 mRNA expression was induced in this cell line by IL-1. Antibody against IL-6 did not neutralize the antiproliferative effect of IL-1. Therefore, for MDA-MB-415 cells IL-6 appeared not to be involved in the antiproliferative effect of IL-1. These results indicate that the antiproliferative effect of IL-1 involves at least two pathways, one IL-6 dependent and another IL-6 independent. The contribution of IL-6 to the antiproliferative effect of TNF was also examined. IL-6 appeared not to play a role in the antiproliferative effect of TNF in these cell lines.     

佛波酯诱导内皮素和FOS／JUN基因在血管内皮细胞中的表达及AP－1结合活性

佛波酯诱导内皮素和ＦＯＳ／ＪＵＮ基因在血管内皮细胞中的表达及ＡＰ－１结合活性温进坤,魏素珍（河北医学院生化教研室,石家庄０５００１７）张晨晖,姚阿卿,周爱儒,汤健（北京医科大学心血管基础研究所,北京,１０００８３）关键词内皮素基因表达;ＡＰ－１转录因...