Molecular analysis of cell cycle-related gene expression in anti-HLA class I monoclonal antibody (01.65) treated PHA-activated human T-lymphocytes.

HLA class I antigens seem to be involved in the proliferative response of PHA-activated human T-lymphocytes. We have previously reported that the treatment of PHA-activated peripheral blood mononuclear cells (PBMC) with an anti-HLA class I monoclonal antibody, 01.65, (i) inhibits the tritiated thymidine incorporation, (ii) inactivates cytosolic protein kinase C (PKC) and (iii) causes an increase in the duration of the cell cycle. Northern Blot kinetic analysis of c-fos, c-myc, cdc2, IL-2R, c-myb, ODC, TK and H3, from 10 minutes to 120 hours, was performed in MAb 01.65 treated cultures. We found that the expression of four genes (c-myc, IL-2R, cdc2 and TK) was depressed 24 hours after PHA stimulation.     

Inhibition by anti-HLA class II monoclonal antibodies of monoclonal antibody OKT3-induced T cell proliferation. Studies at the mRNA level

mAb to monomorphic determinants of HLA class II Ag have been shown to inhibit monocyte-dependent OKT3-induced T cell proliferation, indicating that MHC class II molecules play a regulatory role also in Ag nonrestricted, CD3-induced T cell proliferation. This effect involves several steps in the process of T cell activation and proliferation, including IL-1 beta, IL-6, and IL-2 secretion and IL-2R alpha expression. In the present study, we analyzed the effect of an anti-HLA class II mAb (Q5/6) on the mRNA expression of genes related to monocyte and T cell activation. mRNA levels for early (early c-myc, c-fos) and late (late c-myc, N-ras, c-myb) genes involved in T cell activation were determined as well as mRNA levels for IL-1 beta, IL-6, IFN-gamma, IL-2, and IL-2R alpha. The kinetics of mRNA induction for ICAM-1 was also investigated. The results show that in T lymphocytes the expression of c-fos and early c-myc mRNA was unaffected by mAb Q5/6, whereas the c-myb and N-ras mRNA levels were strongly diminished as well as those of IL-2, IL-2R alpha, and IFN-gamma mRNA. An early increase of ICAM-1 mRNA was partially inhibited. In monocytes, a marked reduction of IL-1 beta and IL-6 mRNA was found. It is concluded that the HLA class II determinant involved in the inhibition mechanism can be engaged in the control of IL-1 beta and IL-6 mRNA levels and constitute an accessory signal up-regulating IL-2 and IL-2R alpha gene activation, through a pathway not affecting c-myc and c-fos expression.     

Requirements for the simultaneous presence of phorbol esters and calcium ionophores in the expression of human T lymphocyte proliferation-related genes

We have investigated the synergistic effects of phorbol ester and calcium ionophore on human T lymphocyte proliferation and the expression of the proliferation-related genes, c-myc, c-fos, interleukin 2 receptors (IL-2R) and interleukin 2 (IL-2). Incubation of T lymphocytes with both the phorbol ester, phorbol 12,13-dibutyrate (PDB), and the calcium ionophore, ionomycin, leads to the expression of a series of proliferation-related genes, followed by T cell proliferation. In contrast, stimulation of T cells sequentially with PDB and then ionomycin did not induce mitogenesis, demonstrating that simultaneous exposure to both agents is necessary for proliferation. Exposure of T cells to both agents together for different time periods resulted in a proliferative response in proportion to the duration of the exposure, with more than 6 hr required for maximum proliferation. In contrast, a 1-hr exposure to both drugs was sufficient for maximum expression of c-fos or c-myc proto-oncogene mRNA. The expression of IL-2R and the production of IL-2 were also dependent on the duration of simultaneous exposure to both phorbol ester and calcium ionophore. Levels of IL-2 mRNA became detectable at 1 hr and peaked at 3 hr after stimulation. The induction of IL-2 mRNA occurred only in the presence of both agents and became undetectable within 2 hr after the drugs were removed. In contrast, the expression of IL-2R mRNA became detectable at 1 hr, but was maintained even after the drugs were removed and reached a peak at 24 hr. Both IL-2 and IL-2R mRNA accumulated in proportion to the duration of the exposure. Augmentation of cell proliferation by exogenous IL-2 was observed in T cells exposed to the drugs for less than 3 hr. These data demonstrated that the induction of maximum expression of the nuclear proto-oncogenes c-myc and c-fos was not sufficient for PDB-ionomycin-induced T cell proliferation. The level of IL-2 mRNA accumulation and resultant IL-2 secretion is one of the limiting factors for proliferation of T cells exposed to the drugs for less than 3 hr, but not for longer exposures. Additional events such as accumulation of IL-2R mRNA and protein triggered by a long exposure to the drugs were obligatory for obtaining maximum proliferation.     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of Ej-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

Transformation of T lymphocytes by the v-fos oncogene

Activation of T lymphocytes through the T cell antigen receptor has been shown to stimulate a rapid and transient accumulation of c-fos mRNA and protein. Transfection of a normal murine T lymphocyte clone with the FBJ-v-fos oncogene resulted in generation of a cell line that was morphologically transformed, had lost the requirement for IL-2 for proliferation, and was tumorigenic in adult syngeneic mice; however, the transformed cells retained the ability to proliferate in response to IL-2. The transformed cells did not show constitutive expression of IL-2 or c-fos mRNA, although the promoter regions of both IL-2 and c-fos genes contain AP-1 sites that are expected to be targets for binding of Fos/Jun complexes. In contrast, the transformed T cells showed increased constitutive expression of IL-2R alpha and c-myc mRNA; these genes may represent cellular targets for transformation by v-fos and physiologic activation by c-fos. We discuss the possibility that these transformed cells behave as cells partially activated through the TCR, and that transformation occurs through a mechanism independent of IL-2.     

A burst of c-fos gene expression in the mouse occurs at birth.

Expression of the c-fos gene during murine perinatal development was studied. Before birth, all eight of the prenatal organs tested expressed undetectable or low levels of c-fos mRNA. On the day of birth, there occurred a 10- to 100-fold increase in the level of c-fos message in all of these organs. The expression was transient, in that 1 day after birth, the level of c-fos mRNA precipitously dropped. The c-fos gene expression at birth is unrelated to the expression of the c-myc gene and major histocompatibility complex class I genes, which display distinct kinetics during the perinatal development. The c-fos gene was also expressed locally and transiently in the gravid uterus 1 to 2 days prior to delivery. These results indicate that an event associated with birth induced c-fos gene expression in the mother and newborn.     

Intereukin-10 and Kupffer cells protect steatotic mice livers from ischemia-reperfusion injury

Steatotic livers are more sensitive to ischemia/reperfusion (I/R) and are thus routinely rejected for transplantation because of their increased rate of primary nonfunction (PNF). Lean livers have less I/R-induced damage and inflammation due toKupffer cells (KC), which are protective after total, warm, hepatic I/R with associated bowel congestion. This protection has been linked to KC-dependent expression of the potent anti-inflammatory cytokine interleukin-10 (IL-10).We hypothesized that pretreatment with exogenous IL-10would protect the steatotic livers of genetically obese (ob/ob) mice from inflammation and injury induced by I/R. Lean and ob/ob mice were pretreated with either IL-10 or liposomally-encapsulated bisphosphonate clodronate (shown to deplete KC) prior to total, warm, hepatic I/R. IL-10 pretreatment increased survival of ob/ob animals at 24 hrs post-I/R from 30% to 100%, and significantly decreased serum ALT levels. At six hrs post-I/R, IL-10 pretreatment increased IL-10 mRNA expression, but suppressed up-regulation of the pro-inflammatory cytokine IL-1β mRNA. However, ALT levels were elevated at six hrs post-I/R in KC-depleted animals. These data reveal that pretreatment with IL-10 protects steatotic livers undergoing I/R, and that phagocytically active KC retain a hepatoprotective role in the steatotic environment.     

Increased levels of c-fos and c-myc mRNA in ATP-stimulated endothelial cells

In bovine aortic endothelial cells, ATP induced a transient and sequential accumulation of c-fos and c-myc mRNA, which was detected after 1 hour and 3 hours, respectively. The effect of ATP on c-fos mRNA was stronger than that of TNF and bFGF. Both ATP and bFGF increased c-myc mRNA after a 3 hour treatment, whereas TNF did not. If none of the 3 agonists tested induced a selective expression of c-fos or c-myc, each of them was associated with a different quantitative combination of the 2 signals, which might be related to the distinct responses that they trigger in endothelial cells.     

Modification of proto-oncogene expression by phorbol esters in human dermal microvascular endothelial cells.

Following activation with the inflammatory mediator phorbol myristate acetate (PMA), human microvascular endothelial cells (DMEC) is olated from the human dermis (DMEC) rapidly and dramatically convert from a classical epithelioid morphology to a spindle-shaped configuration. This is accompanied by changes in the organization of gap junctions and the vimentin and actin cytoskeletons. This report describes the sequential changes in the expression of four proto-oncogenes, c-fos, c-myc, c-sis and H-ras in DMEC following PMA exposure. The synthesis of c-fos mRNA was transiently induced by PMA from a basal concentration below the limit of detection to a maximum at 60 min., declining to the unstimulated level within 2 hrs. Synthesis of c-myc mRNA declined continuously and reached 37% of control levels over 16 hrs. Expression of c-sis which encodes for the B chain of platelet-derived growth factor, also declined to 34% of the control value over 16 hrs. There was no change in the synthesis of H-ras mRNA nor of beta-actin mRNA which was used as a control. The expression of c-myc in normal DMEC was compared to a human dermal microvascular cell line transformed by SV-40 (TREND). The TREND cell line maintains a permanent spindle-shaped configuration under all growth conditions and multiplies faster than DMEC. In contrast to the non-transformed cell cultures, expression of c-myc in TREND cells was induced by PMA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bombesin induction of c-fos and c-myc proto-oncogenes in Swiss 3T3 cells: significance for the mitogenic response

Bombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c-fos and c-myc mRNAs. Enhanced expression of c-fos (122 +/- 14-fold) occurred within minutes of peptide addition followed by increased expression of c-myc (82 +/- 16-fold). The concentrations of peptide required for half-maximal increase in the levels of c-fos and c-myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin-stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c-fos and c-myc mRNA levels promoted by bombesin. Down-regulation of protein kinase C by long-term exposure to phorbol esters prevented c-fos and c-myc induction by bombesin. This and other results indicate that the induction of these proto-oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c-fos and c-myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c-fos and c-myc mRNA levels. This apparent dissociation of mitogenesis from proto-oncogene induction was even more dramatic in 3T3 cells with down-regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c-fos and c-myc mRNA levels at comparable concentrations. Thus, the induction of c-fos and c-myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto-oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.     

外源性神经节苷脂GM_3影响人肝癌细胞生长的可能机制的初步研究

通过苔酚蓝染色细胞发现,外源性GM3（10μg/ml）能明显抑制人肝癌细胞株SMMC-7721细胞生长,在GM3处理3d时,出现明显差异．通过NorthernBlot分析发现,外源性GM3可明显影响人肝癌细胞株SMMC-7721细胞中c-fos、c-jun、c-myc和N-ras这四种癌基因的mRAN表达．未经GM3处理的细胞中没有检测到c-fosmRNA,但c-jun微量表达,并有c-myc和N-rasmRNA的高水平表达而GM3可在短时间内快速大量地诱导c-fos、c-junmRNA的生成．GM3处理的细胞,c-myc和N-rasmRNA的表达均明显减少．GM3处理45min时,c-myc基因表达只为对照组的39．55％;GM3处理24h时,N-ras基因表达为对照组的30.48％．以上结果提示：GM3抑制SMMC-7721细胞生长很可能是通过改变癌基因表达来实现的．     

Anti-CD3 antibody induces rapid expression of cytokine genes in vivo

Anti-CD3 antibody was administered to mice i.v. and the kinetics of spleen cell cytokine mRNA expression studied by Northern analysis. Untreated mice and mice receiving control antibody had low or undetectable amounts of mRNA for c-fos, c-myc, IL-2, IL-4, and IFN-gamma. After injection of anti-CD3 antibody, substantial increases in all were found. Induction of c-fos was detected at 10 min and of c-myc at 30 min after injection. IL-2, IL-4, and IFN-gamma mRNA were induced by 30 min and reached peak levels at 60 min. Thereafter, IL-2 and IL-4 mRNA declined, whereas IFN-gamma mRNA persisted. The induced cytokine mRNA was not observed in athymic nu/nu mice nor in normal spleen cells from which T cells had been depleted in vitro. The early in vivo induction of IL-4 mRNA contrasts with prior in vitro studies in which IL-4 production was difficult to detect after primary stimulation. To assess the possibility that many T cells had been preprimed in vivo, germfree mice were compared with conventional mice and no differences in cytokine mRNA were found. These data show that T cell-dependent IL message production can be induced rapidly in vivo without prepriming and that the cytokine messages induced after anti-CD3 antibody administration do not suggest a predominance of either Th1 or Th2 type cells.     

Effects of epidermal growth factor and platelet-derived growth factor on c-fos and c-myc mRNA levels in normal human fibroblasts

The mRNA levels of two proto-oncogenes, c-fos and c-myc, were determined in human foreskin fibroblasts exposed to epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) in a serum-free, defined medium (MCDB 104). Untreated, quiescent cells were found to have low or undetectable levels of c-fos and c-myc mRNA. Within 10 min after the addition of EGF or PDGF the c-fos mRNA level increased, reached a peak at 30 min, and then declined to the control level after 60 min. The level of c-myc mRNA increased somewhat later and peaked after 8 h in cultures treated with either of the growth factors. The c-myc mRNA level remained elevated throughout the 24 h of investigation. The concentrations of EGF and PDGF required for a maximal effect on c-fos or c-myc expression were found to be similar to those that give maximal effect on cell proliferation. Both c-fos and c-myc mRNA expression were super-induced by the addition of cycloheximide. The addition of neutralizing PDGF antibodies to cultures that had received PDGF 4 h earlier inhibited the subsequent increase in the c-myc mRNA level, indicating that the effect of PDGF on c-myc expression is not caused by a "hit and run," mechanism. Density-inhibited cells responded to EGF and PDGF by an increase in c-fos and c-myc mRNA levels in the absence of any mitogenic response. The present results conform to the view that the c-fos and c-myc proto-oncogenes may be important (or necessary) but not sufficient for the initiation of DNA synthesis. Moreover, the finding that both EGF and PDGF increase c-fos and c-myc expression supports our previous suggestion that these two growth factors may in part act via a common intracellular pathway in the prereplicative phase of human fibroblasts.     

Bombesin induces c-fos and c-myc expression in quiescent Swiss 3T3 cells. Comparative study with other mitogens

We have studied the effect of the potent mitogen bombesin on the expression of c-fos and c-myc genes in quiescent mouse fibroblasts. We have demonstrated that bombesin rapidly induces a transient expression of c-fos mRNA followed by a more protracted elevation in c-myc mRNA levels. The intensity of the induction of expression of both proto-oncogenes depended on the dose of bombesin used. Prolonged treatment of the cells with TPA, which causes a selective decrease in protein kinase C activity, partially inhibited the induction of c-fos and c-myc gene expression by bombesin, similar to what has been observed with PDGF. However, a dramatic inhibition of the mitogenic response to bombesin--but not to PDGF--was found in TPA-treated cells. In contrast, TPA-treated cells showed an increased response to EGF with regard to proto-oncogene expression. The role of protein kinase C and Ca2+-dependent pathways in proto-oncogene induction by bombesin is discussed.     

Activation of 'immediate-early' genes by estrogen is not sufficient to achieve stimulation of DNA synthesis in rat uterus

17 beta-estradiol, a long acting estrogen that is mitogenic for rat uterus in vivo, or the short acting estrogens estriol and 16 alpha-estradiol, not mitogenic on their own, were injected into adult, castrated rats and their effect on uterine gene expression and rate of DNA synthesis were compared. All three compounds increased steady-state mRNA concentration of c-fos, c-jun and c-myc proto-oncogenes to comparable levels (2 hrs after treatment), whereas only 17 beta-estradiol was found to stimulate significantly DNA synthesis (20-22 hrs later). Based on the different retention time of the tested estrogens in rat tissues, it is concluded that a short exposure to the hormone is sufficient to render uterine cells competent to progress through the cell cycle, via activation of 'immediate-early' genes expression, but that stimulation of DNA synthesis requires further changes, achieved via a prolonged exposure of the cells to the estrogenic stimulus.