Thyrotropin and dibutyryl cyclic AMP increase levels of c-myc and c-fos mRNAs in cultured rat thyroid cells

We have studied the effects of thyrotropin (TSH) on the growth and on the levels of the mRNAs of the cellular proto-oncogenes, c-myc, and c-fos, in the specific target of TSH action, the thyroid follicular cell. FRTL5 cells, a cloned line from normal rat thyroid gland that depends upon TSH for its replication, were maintained in a quiescent state for 5 days by keeping them in a medium devoid of serum or TSH. The addition of bovine TSH (bTSH, 1 nM) increased DNA synthesis and stimulated cell proliferation after a lag period of 24 h. This growth response was anteceded by prompt, but transient, increases in the levels of c-myc and c-fos mRNAs, with peak responses at 60 and 30 min, respectively. The minimally and maximally effective concentrations of bTSH were 0.01 mM and 1.0 nM, respectively. Dibutyryl cAMP (Bt2cAMP) stimulated cell growth and increased the level of c-myc mRNA in a concentration-dependent manner, with maximum effects at a Bt2cAMP concentration of 1 mM. At the single concentration tested (1 mM), Bt2cAMP also increased the level of c-fos mRNA. Hence, bTSH-stimulated mitogenesis in quiescent FRTL5 cells is associated with rapid, but short-lived, increases in the levels of the mRNAs of the proto-oncogenes, c-myc and c-fos. Since bTSH is known to stimulate adenylate cyclase in these cells, and since the effect of TSH on c-myc and c-fos mRNAs is mimicked by Bt2cAMP, it is possible that these responses to bTSH are mediated, at least in part, by cAMP.     

Endothelin-1 stimulates c-fos mRNA expression and acts as a modulator on cell proliferation of rat FRTL5 thyroid cells.

In FRTL5 thyroid cells, endothelin (ET)-1 alone had no effect on DNA synthesis but caused a transient increase in c-fos mRNA levels and stimulated IGF-I induced DNA synthesis and cell proliferation. By contrast, ET-1 inhibited the stimulatory effects of TSH actions on DNA synthesis, cell proliferation and c-AMP production. 8-Bromo-cAMP-induced DNA synthesis was also inhibited by ET-1, suggesting that ET-1 exerts its inhibitory effects at step(s) involving cAMP production and post cAMP pathway. ET-1-induced suppression of TSH actions were reversed by a C-kinase inhibitor, H-7. These results suggest that the effect of ET on functions of FRTL5 cells is, at least, in part mediated by C-kinase dependent pathway.     

Heregulin-dependent autocrine loop regulates growth of K-ras but not erbB-2 transformed rat thyroid epithelial cells

The EGF-like family of proteins, such as epidermal growth factor (EGF), transforming growth factor α (TGFα), amphiregulin (AR), betacellulin (BTC), cripto-1 (CR-1), and heregulin (HRG), plays an important role in the pathogenesis of several human carcinomas as autocrine growth factors. Differentiation and proliferation of rat thyroid cells in culture (FRTL-5 cells) are regulated by thyrotropin (TSH); withdrawal of TSH from culture medium produces growth arrest, whereas its addition to quiescent cells stimulates cell entry into S phase. Instead, transformed thyroid cell lines as FRTL-5H2 cell line, overexpressing erbB-2, Kimol cells, transformed by the wild-type K-ras and A6 clone, transformed by a temperature sensitive K-ras mutant, can grow without addition of TSH to the culture medium. In order to identify whether EGF-like growth factors and corresponding receptors (erbB-2, erbB-3, and erbB-4) could be involved in the autonomous growth of these transformed rat thyroid epithelial cells, Northern blot for mRNA analysis and Western blot for protein expression were performed. In contrast to normal control FRTL-5 cells, both K-ras and erbB-2-transformed cells expressed elevated levels of erbB-2 receptor. Moreover, both K-ras transformed cells, Kimol and A6 cells, but no FRTL-5H2 cells, were found able to express also high levels of erbB-4 receptor and HRG/NDF ligand. Treatment of K-ras transformed thyroid cells with neutralizing antibody against HRG/NDF reduced by 50% cell proliferation. These data indicate that unlike the erbB-2 overexpressing FRTL-5 cells, in K-ras rat thyroid epithelial cells, the growth factor heregulin signals through the heterodimer erbB-2/erbB-4 receptors in an autocrine fashion. J. Cell. Physiol. 176:383–391, 1998. © 1998 Wiley-Liss, Inc.     

Production of urokinase-type plasminogen activator by normal and transformed rat thyroid cells in culture

We studied the relationship between differentiation, transformation, and uPA production in a system of rat thyroid cells in vitro. The fully differentiated FRTL5 cells did not produce detectable amounts of uPA, even after stimulation with phorbol esters, potent inducers of uPA expression. All the other cell lines (i.e., FRT, cells which have lost the characteristics of the differentiated thyroid cells; 1-5 G and FRA, transformed cells derived from rat thyroid tumors) produced uPA, the 1-5 G line being the highest producer. Also the FRTL line became positive for uPA production after viral transformation (clone KM4). The lack of uPA expression in FRTL5 cells was not due to the presence of inhibitors and these cells did not produce an inactive molecule, as shown by immunoprecipitation with anti-uPA antibody. However, in FRTL5 cells Northern analysis showed the presence of a small amount of uPA-specific mRNA that increased appreciably after phorbol ester stimulation. In conclusion, in our system uPA expression was a property of undifferentiated and transformed cells; in fully differentiated cells uPA expression was switched off by a still unclear mechanism.     

A novel TNF-inducible message with putative growth suppressor function

We report the nucleotide sequence of a novel cDNA and TNF-induced expression of the corresponding message (mRNA) in human fibroblast cells. This message is also expressed in certain human tumor cell lines and is over-expressed in a colon cancer cell line (HT-29). NIH3T3 cells transfected with the antisense construct of the 5'-region of this novel cDNA formed 20-fold more colonies in culture compared to cells transfected with a sense construct of the same region or the sense and the antisense constructs of the central region of this cDNA. This observation suggests a possible growth suppressor function for the gene represented by this cDNA.     

Growth-Regulatory Activity of the Growth Arrest-Specific Gene, GAS1, in NIH3T3 Fibroblasts

The growth arrest-specific gene, Gas-1, is preferentially expressed in quiescent NIH3T3 cells and inhibits DNA synthesis, suggesting that Gas-1 may be a tumor suppressor gene. When GAS1 cDNA, under the control of the strong constitutive CMV promoter, was transfected into NIH3T3 cells, no stable transfectant cell lines were produced, confirming that high levels of expression of GAS1 mRNA inhibit proliferation. GAS1, under the control of a dexamethasone-inducible promoter, was also transfected into NIH3T3 cells, resulting in normal numbers of transfectant clones. When expression of GAS1 mRNA was induced with dexamethasone, the growth rate was greatly inhibited. Morphological changes characteristic of growth arrest were also observed. To determine if antisense inhibition of expression of Gas-1 will transform normal fibroblasts, GAS1 cDNA, cloned in the antisense orientation, was transfected into NIH3T3 cells and expression of endogenous Gas-1 mRNA was inhibited. The GAS1-antisense cells had altered morphology and grew to a much higher saturation density than control cell lines with a loss of contact inhibition. However, there was no change in requirements for serum or any development of anchorage-independence. Antisense inhibition of expression of GAS1 is therefore insufficient to transform the cells, suggesting that additional genetic events are required for a fully malignant phenotype.     

Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.     

Somatostatin interferes with thyrotropin-induced G1-S transition mediated by cAMP-dependent protein kinase and phosphatidylinositol 3-kinase. Involvement of RhoA and cyclin E x cyclin-dependent kinase 2 complexes

cAMP-mediated cell proliferation is a complex process that involves multiple pathways. Using a cAMP-dependent cell system, FRTL-5 thyroid cells, we have previously demonstrated the existence of a precise autocrine loop in the control of cell proliferation that involves the positive effector thyrotropin (TSH) and the general inhibitor somatostatin. In search of the regulatory mechanisms responsible for the TSH and somatostatin control of cell proliferation, we analyzed the cell cycle regulatory proteins and the cellular pathways involved in the action of both signals. The results show that specific inhibition of cAMP-dependent protein kinase (PKA) and phosphatidylinositol (PI) 3-kinase blocks independently TSH-induced FRTL-5 cell proliferation and that somatostatin interferes with both signals. Each pathway activates different proteins required for G(1)/S progression. Thus, PKA is responsible for the TSH-induction of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels, RhoA activation, and down-regulation of p27(kip1). These correlated events are necessary for FRTL-5 cell proliferation after TSH stimulation. Moreover, TSH through PKA pathway increases cyclin-dependent kinase 2 levels, whereas PI 3-kinase signaling increases cyclin E levels. Together, both pathways finally converge, increasing the formation and activation of cyclin E x cyclin-dependent kinase 2 complexes and the phosphorylation of the retinoblastoma protein, two important steps in the transition from G(1) to S phase in growth-stimulated cells. Somatostatin exerts its antiproliferative effect inhibiting more upstream the TSH stimulation of PKA and PI 3-kinase, interfering with the TSH-mediated increases of intracellular cAMP levels by inactivation of adenylyl cyclase activity. Together, these results suggest the existence of a PKA-dependent pathway and a new PKA-independent PI 3-kinase pathway in the TSH/cAMP-mediated proliferation of FRTL-5 thyroid cells.     

Overexpression of phospholipase C-gamma1 suppresses UVC-induced apoptosis through inhibition of c-fos accumulation and c-Jun N-terminal kinase activation in PC12 cells.

Ultraviolet-C (UVC) irradiation induces DNA damage and UVC-irradiated cells undergo cell growth arrest to repair the damaged DNA or the induction of apoptosis to prevent the risk of neoplastic transformation. Phospholipase C-gamma1 (PLC-gamma1) is a mediator of growth factor induced-signal cascade, catalyzing the hydrolysis of phosphatidyl 4,5-bisphosphate to generate second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)). PLC-gamma1 is activated by phosphorylation of tyrosine residues upon occupation of cell surface receptors by growth factors and plays an important role in controlling cellular proliferation and differentiation. In this study, we found that PLC-gamma1 was tyrosine phosphorylated within 2.5 min after UVC irradiation. To investigate the role of UVC-induced tyrosine phosphorylation of PLC-gamma1, we compared the effect of UVC between PLC-gamma1 overexpressing cells and empty vector transfected cells. Overexpression of PLC-gamma1 inhibited UVC-induced sub-diploid peak and DNA fragmentation. Northern blot analysis revealed that UVC-induced c-fos mRNA accumulation was inhibited in PLC-gamma1 overexpressing cells, while c-jun expression was not affected. In addition, UVC-induced activation of c-Jun N-terminal kinase (JNK) was significantly suppressed in PLC-gamma1 overexpressing cells. These results suggest that PLC-gamma1 may associate with the protective function against the UVC-induced cell death progression via the inhibition of accumulation of c-fos mRNA and the inhibition of JNK kinase activity.     

Studies on the promoter region of the c-Ha-ras gene in FRTL5 rat thyroid cells

The functional activity of the promoter region of the rat c-Ha-ras gene was examined in FRTL5 rat thyroid cells, the cell type from which this promoter was cloned. A plasmid (p035-ras-CAT) was constructed containing the untranslated-1 exon as well as 172 base pairs (bp)5' to this exon inserted upstream of the chloramphenicol acetyl transferase (CAT) reporter gene. These 172 bp of 5'-flanking region contain two 10 bp GC box consensus sites and two CAAT boxes. Very weak promoter activity was observed in experiments involving transient transfection of FRTL5 cells with this plasmid, as well as with another plasmid (p5kb-ras-CAT) containing a much more extensive (3.5 kb) 5'-flanking region of the gene. In contrast, strong promoter activity was observed when the same plasmids were transfected into mouse 3T3 fibroblasts. When other promoters (pfos, RSV, and MMTV) were used to drive CAT activity, CAT activity in FRTL5 cells was about 10-fold less than in NIH-3T3 cells and rat embryo fibroblasts. However c-Ha-ras promoter activity was reduced out of proportion in FRTL5 thyroid cells relative to the other cell types (approximately 50-fold less). DNA gel-shift assays performed using crude extracts of FRTL5 and 3T3 nuclear proteins revealed quantitatively similar binding to the same promoter region in the c-Ha-ras 5'-flanking sequence. These data demonstrate that promoter activity of the rat c-Ha-ras gene is contained within the 172 bp 5'-flanking region of the gene. This promoter activity is expressed at a much lower level in slow-growing FRTL5 cells relative to other more rapidly growing cell types.     

Effects of basic fibroblast growth factor on angiogenin expression and cell proliferation in H7402 human hepatoma cells

Hepatocellular carcinoma （HCC） is one of the most common cancers worldwide. Basic fibroblast growth factor （bFGF）, which is highly expressed in developing tissues and malignant cells, regulates cell growth, differentiation, and migration. Its expression is essential for the progression and metastasis of HCC. This study aims to investigate the effects of bFGF on the expression of angiogenin, another growth factor, which plays an important role in tumor angiogenesis, and on cell proliferation in H7402 human hepatoma cells. The bFGF sense cDNA or antisense cDNA was stably transfected into H7402 cells. Genomic DNA PCR analysis demonstrated that human bFGF sense cDNA or antisense cDNA was inserted into the genome. Furthermore, the expression of bFGF and angiogenin was examined by RT-PCR and Western blot assays. MTT and colony formation assays were employed to determine cell proliferation. Stable bFGF over-expressing and under-expressing transfectants were successfully established. Expression of angiogenin was decreased in the over-expressing bFGF cells （sense transfectants） and was increased in the under-expressing bFGF cells （antisense transfectants）. Cell proliferation increased in the bFGF sense transfectants and decreased in the bFGF antisense transfectants. These results demonstrated that the endogenous bFGF may not only negatively regulate the angiogenin expression but also contribute to the overall cell proliferation in H7402 human hepatoma cells. This study may be helpful in finding a potential therapeutic approach to HCC.     

Purinergic agonist ATP is a comitogen in thyroid FRTL-5 cells

Several growth factors may stimulate proliferation of thyroid cells. This effect has, in part, been dependent on calcium entry. In the present study using FRTL-5 cells, we show that in addition to its effect on calcium fluxes, ATP acts as a comitogen in these cells. In medium containing 5% serum, but no TSH, ATP stimulated the incorporation of ³H-thymidine in a dose- and time-dependent manner in the cells. At least a 24-h incubation with ATP was necessary to observe the enhanced (30–50%) incorporation of ³H-thymidine and an increased (30%) cell number. The effect of ATP was dependent on insulin in the incubation medium. Furthermore, ATP enhanced the TSH-mediated incorporation of ³H-thymidine. The effect of ATP was apparently mediated via a G-protein dependent mechanism, as no stimulation of thymidine incorporation was observed in cells treated with pertussis toxin. The effect of ATP was not dependent on the activation of protein kinase C (PKC), as ATP was effective in cells with downregulated PKC. ATP rapidly phosphorylated mitogen activated protein (MAP) kinase in FRTL-5 cells. In addition, ATP stimulated the expression of a 62 kDa c-fos dependent protein in a dose- and time-dependent manner. Our results thus suggest that extracellular ATP, in the presence of insulin, may be a cofactor in the regulation of thyroid cell proliferation, probably by phosphorylating MAP kinase and stimulating the expression of c-fos. © 1996 Wiley-Liss, Inc.     

Specific down-regulation of annexin II expression in human cells interferes with cell proliferation

The protein-tyrosine kinase substrate annexin II is a growth regulated gene whose expression is increased in several human cancers. While the precise function of this protein is not understood, annexin II is proposed to be involved in multiple physiological activities, including DNA synthesis and cell proliferation. Targeted disruption of the annexin II gene affects calcium signaling, tyrosine phosphorylation and apoptosis, indicating the important physiological role of this protein. We used a transient co-transfection assay to regulate annexin II expression in human HeLa, 293 and 293T cells, and measured the effects of annexin II down regulation on DNA synthesis and proliferation. Transfection of cells with an antisense annexin II vector results in inhibition of cell division and proliferation, with concomitant reduction in annexin II message and protein levels. Cellular DNA synthesis is significantly reduced in antisense transfected cells. Replication extracts made from antisense transfected cells have significantly reduced efficiency to support SV40 in vitro DNA replication, while the extracts made from sense transfected cells are fully capable of replication. Our results indicate an important role of annexin II in cellular DNA synthesis and cell proliferation.     

桦木酸对人胃癌SGC-7901细胞增殖的影响*

目的:利用不同浓度的桦木酸对人胃癌SGC-7901细胞增殖的影响。方法:桦木酸设4个不同浓度(0、10、20、30 μg/ml),并采用常规化疗药物5-Fu处理作为阳性对照,以探究其对细胞增殖的影响。采用台盼蓝拒染法和吉姆萨染色法分别检测桦木酸对人胃癌SGC-7901细胞生长抑制率及克隆形成率;EdU法检测SGC-7901的细胞增殖;利用流式细胞术检测细胞周期, 应用qRT-PCR和Western blot分别检测细胞周期蛋白cyclin D1,cyclin B1的mRNA和蛋白表达水平。结果:不同浓度的桦木酸处理人胃癌SGC-7901细胞48 h后,其细胞生长抑制率显著升高(P＜0.05),克隆形成率和细胞增殖率均明显降低(P＜0.01),且呈剂量和时间依赖性;人胃癌SGC-7901细胞被阻滞在G1/G0期,细胞周期蛋白cyclin D1和cyclin B1的mRNA和蛋白表达量也随桦木酸浓度升高而显著降低(P＜0.01)。且与5-Fu对照组相比,桦木酸浓度为20 μg/ml和30 μg/ml时,细胞增殖能力明显降低,细胞周期被抑制,细胞周期蛋白表达量均明显降低(P ＜0.05)。结论:桦木酸通过下调cyclin B1和cyclin D1基因表达,将人胃癌SGC-7901细胞阻滞在G1/G0期,从而抑制细胞增殖。