Increase of MnSOD expression and decrease of JNK activity determine the TNF sensitivity in bcl2-transfected L929 cells.

To investigate the protection mechanism of Bcl-2 against tumour necrosis factor (TNF)-mediated cell death, the bcl2 gene was transfected into the L929 cells and stably expressed. Two clones having different sensitivity among bcl2-transfected L929 clones had been isolated, and termed clone R1 and R2. It was observed that activation of manganese superoxide dismutase (MnSOD) and suppression of Jun kinase of clone R1 and R2 were correlated with protection from TNF cytotoxicity. Upon treatment with TNF, clone R1 and R2 were more resistant than control L929 cells against TNF cytotoxicity and the protective effect of clone R1 was stronger than clone R2. However, in case of TNF plus actinomycin D treatment, clone R1 was still resistant against TNF cytotoxicity, whereas clone R2 became more sensitive than control L929 cells. The JNK activities of clone R1 and R2 were suppressed upon TNF treatment and in case of TNF plus actinomycin D treatment, clone R2 showed a marked increase in JNK activities and had higher activity than control L929 cells. The specific activities of MnSOD of clone R1 and R2 upon TNF treatment were 70 U/ml and 33 U/ml, respectively, while the MnSOD activity was not detectable in control L929 cells. When TNF and actinomycin D were treated simultaneously, MnSOD activity was not detectable in control L929 cells and bcl2 -transfected L929 cells (clone R1, R2). Consistent with these results, both clone R1 and R2 showed higher levels of MnSOD mRNA expression than control L929 cells after TNF treatment. These data suggest that suppression of Jun kinase and increase of MnSOD may be involved in inhibitory action of Bcl-2 against TNF, and the balance between MnSOD and JNK signalling pathway may be an important factor for the protection of bcl2-transfected L929 cells from TNF cytotoxicity.     

Expression of fibroblast growth factor receptor 3 by fibroblast growth factor 2 in cultured chick embryo chondrocytes

Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression.     

Identification and validation of a gene involved in anchorage-independent cell growth control using a library of randomized hairpin ribozymes

We have developed a library of hairpin ribozyme genes that can be delivered and expressed in mammalian cells with the purpose of identifying genes involved in a specific phenotype. By applying the appropriate phenotypic selection criteria in tissue culture, we can enrich for ribozymes that knock down expression of an unknown gene or genes in a particular pathway. Once specific ribozymes are selected, their target binding sequence is used to identify and clone the target gene. We have applied this technology to identify a putative tumor suppressor gene that has been activated in HF cells, a nontransformed revertant of HeLa cells. Using soft agar growth as the selection criteria for gain of transformation, we have isolated ribozymes capable of triggering anchorage-independent growth. Isolation of one of these ribozymes, Rz 568, led to the identification and cloning of the human homologue of the Drosophila gene ppan, a gene involved in DNA replication, cell proliferation, and larval development. This novel human gene, PPAN, was verified as the biologically relevant target of Rz 568 by creating five additional "target validation" ribozymes directed against additional sites in the PPAN mRNA. Rz 568 and all of the target validation ribozymes reduced the level of PPAN mRNA in cells and promoted anchorage-independent growth. Exogenous expression of PPAN in HeLa and A549 tumor cells reduced their ability to grow in soft agar, underscoring its role in regulating anchorage-dependent growth. This study describes a novel method for gene discovery where the intracellular application of hairpin ribozyme libraries was used to identify a novel gene based solely on a phenotype.     

Effect of ribozyme against transforming growth factorbeta1 on biological character of activated HSCs

Transforming growth factorbeta1 (TGFbeta1) is considered to be the principal contributor to liver fibrosis. So in this study the ribozymes against TGFbeta1 were designed. The in vitro cleavage activities of the ribozymes were assayed through incubation of (32)p-labeled target RNAs and (32)p-labeled ribozymes in different conditions. HSC-T6 cells were transfected with the eukaryotic constructs encoding ribozyme and disable ribozyme, then the stable cell clones were used to evaluate its antifibrotic characteristic through the effect of ribozyme on biological character of activated hepatic stellate cells (HSCs). The results demonstrated that two ribozymes (Rz803 and Rz1395) could cleave target RNAs into expected products effectively, Rz803 possessed better cleavage activity in vitro. Stable transfection of Rz803 into activated HSCs reduced TGFbeta1 expression in mRNA and protein level efficiently. The further studies demonstrated that Rz803 reduced deposition of collagen I, suppressed HSC proliferation, but had no effect on HSC activation in transfected HSC-T6 cells. Therefore, it indicated that Rz803 could reverse the character of activated HSCs by down-regulating TGFbeta1 expression efficiently and diminishing TGFbeta1 signaling underlying activation of hepatic stellate cells. As the consequence, it would provide a potential therapeutic approach for liver fibrosis.     

混合Ribozyme联合切割小鼠腺苷脱氨酶mRNA研究

采用计算机辅助设计得到针对小鼠腺苷脱氨酶ｍＲＮＡ的４种ｒｉｂｏｚｙｍｅ,它们均能在各自的切点切割ＲＮＡ分子,４种ｒｉｂｏｚｙｍｅ经组合后成混合ｒｉｂｏｚｙｍｅ,它们分别含有２种、３种和４种ｒｉｂｏｚｙｍｓ。通过对体外转录靶ＲＮＡ分子的联合切割研究表明,随着ｒｉｂｏｚｙｍｅ种数的增加,其中以（Ｒｚ２６２＋Ｒｚ４５５＋Ｒｚ５８３）组成最为理想,由此证明混合ｒｉｂｏｚｙｍｅ中,各种ｒｉｂｏｚｙｍｅ不仅保     

Role and regulation of the fibroblast growth factor axis in human thyroid follicular cells

Thyroidal levels of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) are elevated in human thyroid hyperplasia. To understand the significance of this, effects of FGFR1 activation on normal human thyrocyte growth and function in vitro and the regulation of FGF-2 and FGFR1 expression have been examined. FGF-2 stimulated cell growth, as measured by cell counting, and inhibited thyroid function as measured by 125I uptake. Sensitivity to FGF-2 disappeared after 7 days, although FGFR1 expression was maintained. Thyroid-stimulating hormone (TSH, 300 mU/l) increased FGFR1 mRNA expression within 4 h and protein expression by 8 h. Exogenous FGF-2 decreased FGFR1 protein. Endogenous FGF-2 levels were low (approximately 1-2 pg/microg protein), and TSH treatment decreased these by 50%. Protein kinase C (PKC) activation increased FGF-2 mRNA and FGF-2 secretion within 2 h. This effect was enhanced (4.4-fold) when cells were cultured in TSH. We conclude that TSH stimulates FGFR1 but not FGF-2 expression. PKC activation stimulates FGF-2 synthesis and secretion, and TSH synergizes with PKC activators. Increases in FGFR1 or FGF-2 or in both may contribute to goitrogenesis.     

细胞周期蛋白D1特异性核酶表达载体的构建及其活性

应用mfold程序对锤头状核酶(ribozyme,Rz)和大鼠细胞周期蛋白(cyclin)D1基因的二级结构进行分析,设计合成锤头状Rz基因,通过RT-PCR扩增获得大鼠细胞周期蛋白D1目的基因,将Rz基因和细胞周期蛋白D1基因分别克隆入载体pGEM-3Zf( )中,体外转录Rz基因和靶基因并进行切割实验;将Rz基因与逆转录病毒载体pLXSN重组得到Rz真核表达载体pLXSN-Rz,将其转染入HSC-T6细胞,G418筛选出阳性细胞克隆,用RT-PCR检测细胞周期蛋白D1基因的表达。结果显示:针对目的基因的832位点设计合成了Rz832,成功获得Rz832基因、细胞周期蛋白D1mRNA的体外转录载体pGEM3Zf-Rz832和pGEM3Zf-cD1,经体外转录出Rz832(105nt)及细胞周期蛋白D1mRNA(1079nt)。体外切割实验证实Rz832能够特异性切割细胞周期蛋白D1mRNA,产生1014nt和65nt的切割产物,切割效率为80%。所构建的pLXSN-Rz832经酶切电泳、PCR鉴定显示,插入的Rz832序列大小约为57bp,与预期结果相同,经测序证实Rz832序列正确。转染pLXSN-Rz832的肝星状细胞(hepaticstellatecells,HSCs)细胞周期蛋白D1mRNA的表达受到明显抑制,仅为对照组的42.22%(t=-193.443,P<0.01),结果表明:Rz832能够在体外特异性切割细胞周期蛋白D1mRNA、并在HSC-T6细胞内有效抑制细胞周期蛋白D1基因的表达。     

Cell surface fibroblast growth factor and epidermal growth factor receptors are permanently lost during skeletal muscle terminal differentiation in culture

One characteristic of skeletal muscle differentiation is the conversion of proliferating cells to a population that is irreversibly postmitotic. This developmental change can be induced in vitro by depriving the cultures of specific mitogens such as fibroblast growth factor (FGF). Analysis of cell surface FGF receptor (FGFR) in several adult mouse muscle cell lines and epidermal growth factor receptor (EGFR) in mouse MM14 cells reveals a correlation between receptor loss and the acquisition of a postmitotic phenotype. Quiescent MM14 cells, mitogen-depleted, differentiation-defective MM14 cells, and differentiated BC3H1 muscle cells (a line that fails to become postmitotic upon differentiation) retained their cell surface FGFR. These results indicate that FGFR loss is not associated with either reversible cessation of muscle cell proliferation or biochemical differentiation and thus, further support a correlation between receptor loss and acquisition of a postmitotic phenotype. Comparison of the kinetics for growth factor receptor loss and for commitment of MM14 cells to a postmitotic phenotype reveals that FGFR rises transiently from approximately 700 receptors/cell to a maximum of approximately 2,000 receptors/cell 12 h after FGF removal, when at the same time, greater than 95% of the cells are postmitotic. FGFR levels then decline to undetectable levels by 24 h after FGF removal. During the interval in which FGFR increases and then disappears there is no change in its affinity for FGF. The transient increase in growth factor receptors appears to be due to a decrease in ligand-mediated internalization because EGFR, which undergoes an immediate decline when cultures are deprived of FGF (Lim, R. W., and S. D. Hauschka. 1984. J. Cell Biol. 98:739-747), exhibits a similar transient rise when cultures are grown in media containing both EGF and FGF before switching the cells to media without these added factors. These results indicate that the loss of certain growth factor receptors is a specific phenotype acquired during skeletal muscle differentiation, but they do not resolve whether regulation of FGFR number is causal for initiation of the postmitotic phenotype. A general model is presented in the discussion.     

Pituitary tumor-derived fibroblast growth factor receptor 4 isoform disrupts neural cell-adhesion molecule/N-cadherin signaling to diminish cell adhesiveness: a mechanism underlying pituitary neoplasia

We previously identified pituitary tumor-derived fibroblast growth factor receptor 4 (ptd-FGFR4), an alternatively transcribed N-terminally truncated cytoplasmic receptor isoform. Unlike wild-type FGFR4, ptd-FGFR4 facilitates cell transformation and results in pituitary tumor formation in transgenic mice. To investigate differences in the tumorigenic properties of FGFR4 and ptd-FGFR4, we examined their abilities to modulate cell adhesiveness. Introduction of ptd-FGFR4 into GH4 pituitary cells or NIH 3T3 fibroblasts resulted in significant reduction in cell adhesion to a collagen IV matrix compared with FGFR4- or empty vector-transfected cells. This adhesive difference was evident in the absence or presence of FGF stimulation. Furthermore, treatment with beta1-integrin neutralizing antibody markedly reduced adhesiveness in FGFR4-transfected cells but had little effect on the depressed adhesiveness of ptd-FGFR4-transfected cells. Unlike wild-type FGFR4, ptd-FGFR4 does not associate with neural cell-adhesion molecule (NCAM). Cells expressing FGFR4 demonstrate membranous N-cadherin with a noninvasive growth pattern identical to control GH4 cells when injected into immunodeficient mice. In contrast, ptd-FGFR4-expressing cells develop invasive tumors in vivo with marked loss of N-cadherin that localizes to the cytoplasm. Consistent with these changes, beta-catenin expression was diminished and its interaction with N-cadherin was disrupted in the presence of ptd-FGFR4, but both were intact in the presence of wild-type FGFR4. These data highlight the importance of membrane-anchored FGFR4 in assembling a multiprotein FGFR4 complex with NCAM and N-cadherin playing pivotal functions in maintaining normal cell adhesion. Disruption of distinct NCAM/N-cadherin proadhesive complexes by a tumor-derived FGFR4 isoform provides a novel mechanism beyond ligand independence that explains the pathobiology of proliferative and infiltrative but nonmetastatic neoplasms.     

Thyroid hormone activates fibroblast growth factor receptor-1 in bone

Thyroid hormone (T3) and the T3 receptor (TR) alpha gene are essential for bone development whereas adult hyperthyroidism increases the risk of osteoporotic fracture. We isolated fibroblast growth factor receptor-1 (FGFR1) as a T3-target gene in osteoblasts by subtraction hybridization. FGFR1 mRNA was induced 2- to 3-fold in osteoblasts treated with T3 for 6-48 h, and FGFR1 protein was stimulated 2- to 4-fold. Induction of FGFR1 was independent of mRNA half-life and abolished by actinomycin D and cycloheximide, indicating the involvement of an intermediary protein. Fibroblast growth factor 2 (FGF2) stimulated MAPK in osteoblasts, and pretreatment with T3 for 6 h induced a more rapid response to FGF that was increased in magnitude by 2- to 3-fold. Similarly, T3 enhanced FGF2-activated autophosphorylation of FGFR1, but did not modify FGF2-induced phosphorylation of the docking protein FRS2. These effects were abolished by the FGFR-selective inhibitors PD166866 and PD161570. In situ hybridization analyses of TRalpha-knockout mice, which have impaired ossification and skeletal mineralization, revealed reduced FGFR1 mRNA expression in osteoblasts and osteocytes, whereas T3 failed to stimulate FGFR1 mRNA or enhance FGF2-activated MAPK signaling in TRalpha-null osteoblasts. These findings implicate FGFR1 signaling in T3-dependent bone development and the pathogenesis of skeletal disorders resulting from thyroid disease.     

Expression of vascular endothelial growth factor and basic fibroblast growth factor receptors in lung cancer

OBJECTIVE: To determine the expression of two angiogenic factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor receptors (FGFR), in non-small cell lung carcinoma (NSCLC) in relation to tumor stage (TN0, TN1, TN2) and in association with the expression of p53 protein, a potential suppressor of tumor angiogenesis. STUDY DESIGN: The immunohistochemical (IHC) expression of VEGF and FGFR was examined in paraffin sections of 56 NSCLC in relation to the presence of lymph node metastases and p53 expression. Nodal status of NSCLC determined: 27 tumors, N0; 16, N1; and 13, N2 stage. Semiquantitative analysis with a score corresponding to IHC staining intensity and percentage of positive cells was used. Statistical analysis was performed with the chi 2 test. RESULTS: A significant association was noted between VEGF and FGFR expression in NSCLC. No relation was found between VEGF, FGFR expression and lymph node metastasis or p53 expression. CONCLUSION: We assume that VEGF and FGFR act in a synergistic manner in NSCLC and that their expression is not related to lymph node metastases. Angiogenesis is a very complex phenomenon and heterogeneous within tumors. Also, it is affected by microenviromental factors.     

Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells

We previously reported that serotonin (5-HT) increased glial cell line-derived neurotrophic factor (GDNF) release in a 5-HT₂ receptor (5-HT₂R) and mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK)-dependent manner in rat C6 glioma cells (C6 cells), a model of astrocytes. We herein found that 5-HT-induced rapid ERK phosphorylation was blocked by 5-HT₂R antagonists in C6 cells. We therefore examined 5-HT-induced ERK phosphorylation to reveal the mechanism of 5-HT-induced GDNF mRNA expression. As 5-HT-induced ERK phosphorylation was blocked by inhibitors for Gα_q/11 and fibroblast growth factor receptor (FGFR), but not for second messengers downstream of Gα_q/11, 5-HT₂R-mediated FGFR transactivation was suggested to be involved in the ERK phosphorylation. Although FGFR1 and 2 were functionally expressed in C6 cells, 5-HT selectively phosphorylated FGFR2. Indeed, small interfering RNA for FGFR2, but not for FGFR1, blocked 5-HT-induced ERK phosphorylation. As Src family tyrosine kinase inhibitors and microtubule depolymerizing agents blocked 5-HT-induced FGFR2 phosphorylation, Src family tyrosine kinase and stabilized microtubules were suggested to act upstream of FGFR2. Finally, 5-HT-induced GDNF mRNA expression was also inhibited by the blockade of 5-HT₂R, FGFR, and Src family tyrosine kinase. In conclusion, our findings suggest that 5-HT induces GDNF mRNA expression via 5-HT₂R-mediated FGFR2 transactivation in C6 cells.     

Fibroblast growth factor inhibits chondrocytic growth through induction of p21 and subsequent inactivation of cyclin E-Cdk2

Fibroblast growth factor (FGF) and its receptor (FGFR) are thought to be negative regulators of chondrocytic growth, as exemplified by achondroplasia and related chondrodysplasias, which are caused by constitutively active mutations in FGFR3. To understand the growth-inhibitory mechanisms of FGF, we analyzed the effects of FGF2 on cell cycle-regulating molecules in chondrocytes. FGF2 dramatically inhibited proliferation of rat chondrosarcoma (RCS) cells and arrested their cell cycle at the G(1) phase. FGF2 increased p21 expression in RCS cells, which assembled with the cyclin E-Cdk2 complexes, although the expression of neither cyclin E nor Cdk2 increased. In addition, the kinase activity of immunoprecipitated cyclin E or Cdk2, assessed with retinoblastoma protein (pRb) as substrate, was dramatically reduced by FGF-2. Moreover, FGF2 shifted pRb to its underphosphorylated, active form in RCS cells. FGF2 not only induced p21 protein expression in proliferating chondrocytes in mouse fetal limbs cultured in vitro but also decreased their proliferation as assessed by the expression of histone H4 mRNA, a marker for cells in S phase. Furthermore, inhibitory effects of FGF2 on chondrocytic proliferation were partially reduced in p21-null limbs, compared with those in wild-type limbs in vitro. Taken together, FGF's growth inhibitory effects of chondrocytes appear to be mediated at least partially through p21 induction and the subsequent inactivation of cyclin E-Cdk2 and activation of pRb.     

Transforming potential of alternatively spliced variants of fibroblast growth factor receptor 2 in human mammary epithelial cells

A breast cancer cell line developed in our laboratory (SUM-52PE) has a 12-fold amplification and high-level overexpression of the oncogene fibroblast growth factor receptor 2 (FGFR2). Previously, nine different alternatively spliced FGFR2 variants were isolated from this cell line. Overexpression of two variants that differ only in their carboxyl termini (C1 and C3) has been successfully accomplished in the immortalized human mammary epithelial cell line H16N2. FGFR2 expression led to the activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling cascades. Phosphorylation of the adapter protein FGF receptor substrate 2 is much more robust in the cells expressing the C3 variant of FGFR2 compared with the C1 variant. H16N2 cells expressing the full-length FGFR2 with the C1 or C3 carboxyl terminus were tested for their ability to grow under epidermal growth factor (EGF)-independent conditions, in soft agar, and for their ability to invade naturally occurring basement membranes and compared with the parental SUM-52PE cell line. All three cell lines grew under EGF-independent conditions and all were inhibited by the FGFR family specific inhibitor PD173074. The full-length FGFR2-C1 and FGFR2-C3 variants grew robustly in soft agar similar to the parental cell line SUM-52PE. However, cells expressing the C3 variant formed large colonies in agar in both insulin-free and EGF-free medium, whereas the cells expressing the C1 variant required insulin for growth. Soft agar growth was also inhibited by PD173074. Because SUM-52PE was developed from a metastatic breast carcinoma, the FGFR2-overexpressing cell lines were assessed for their ability to invade sea urchin embryo cell membranes. H16N2 cells expressing the C1 carboxyl terminus failed to invade sea urchin embryo cell membranes. By contrast, FGFR2-C3-expressing cells were as invasive as the SUM-52 breast cancer cells and erbB-2-overexpressing H16N2 cells. These results indicate that FGFR2 is a transforming oncogene in human mammary epithelial cells when expressed to levels similar to that found in breast cancer cells with FGFR2 gene amplification. Furthermore, the results suggest that different splice variants have differing transforming activities and that signaling from variants expressing the C3 carboxyl terminus results in more autonomous signaling, cell growth, and invasion.     

双位点核酶对乙型肝炎病毒C基因体外转录物的剪切作用

为探讨双位点核酶对乙型肝炎病毒C基因体外转录物的剪切作用,观察双位点核酶对单一核酶体外剪切的增强作用,同时比较串联核酶和混合核酶的体外切割作用,构建了核酶Rz1,Rz3, Rz1和Rz3的串联核酶(Rz13)体外转录载体, 经体外转录后切割靶RNA. 结果表明:双位点核酶,无论是串联或混合核酶均可增强单一核酶的体外切割作用, 串联和混合核酶中的单一核酶可独立发挥作用;当串联和混合数目为2个时,两者的切割效率差别不大(P＞0.05).