Lipid signalling pathways in normal and ras-transfected NIH/3T3 cells

The role of ras oncogenes in cellular signalling pathways involving phospholipid breakdown was studied in untransfected and proto-H-ras and mutated H-, K- and N-ras transfected NIH/3T3 cells. When the cells were grown at low cell densities, all of the ras transfected cells had 2-4 fold higher diacylglycerol (DAG) levels compared to growing NIH/3T3 cells. At high cell densities, DAG levels decreased in the former and increased in contact inhibited NIH/3T3 cells. In this regard, only cells transformed by mutated cellular and viral H-ras oncogenes (but not by the H-ras proto-oncogene) had elevated DAG levels compared to contact inhibited NIH/3T3 cells. The basal levels of inositol phosphates in ras transfected cells were not significantly different from NIH/3T3 cells and did not vary with cell density. Thus, the elevated DAG levels are not a consequence of increased phosphoinositide hydrolysis. The latter was stimulated by serum and bombesin only in normal and proto-H-ras transfected cells. In contrast, stimulation by bradykinin was observed only in cells transformed by mutated cellular ras oncogenes. Furthermore, aluminum fluoride stimulated phosphoinositide breakdown in the latter cells indicating that there was no uncoupling of the G protein from phospholipase C. Treatment of ras transfected cells with dibutyryl cyclic AMP (DB-cAMP), which causes an inhibition of growth and a reversal of the transformed morphology, did not alter the basal levels of inositol phosphates, DB-cAMP, however, did lower DAG levels in some of the transformed cell lines, but elevated DAG levels in low density NIH/3T3 cells. These findings indicate that the ras gene product p21 is not involved in phosphoinositide hydrolysis and that DAG levels do not correlate with cell growth in either normal or ras transfected NIH/3T3 cells. Thus, p21 appears to alter cell growth through mechanism(s) independent of lipid signalling pathways.     

Identification of a PDGF-like mitoattractant produced by NIH/3T3 cells after transformation with SV40

It has previously been shown that fibroblastic cells transformed by SV40 exhibit a reduced requirement for PDGF for growth. In addition, NIH/3T3 cells lose both their chemotactic response to PDGF and specific cell surface binding of PDGF after transformation with SV40. We have now examined whether the SV40 transformed NIH/3T3 cells are producing a factor which acts similarly to PDGF. Our studies indicate that NIH/3T3 cells transformed with SV 40 produce a factor which shares many biological properties with PDGF. We were unable to detect this activity in conditioned media from nontransformed NIH/3T3 cells. The SV40/NIH/3T3 derived factor appears to possess both chemotactic and mitogenic activity for connective tissue cells but not endothelial or epithelial cells. Furthermore, in preliminary studies, this activity competes with 125I-PDGF for binding to smooth muscle cells. The biochemical properties of the SV40/NIH/3T3 derived factor are different from those of PDGF. The SV40 activity appears to reside in a heat labile acidic protein (pI less than 7.0) of MW less than 30,000 whereas PDGF is a heat stable basic protein (pI9.8) of 30,000 MW. Production of this factor may play a role in the decreased serum requirement for cell replication exhibited by SV40-transformed NIH/3T3 cells by supplying the cells with their own PDGF-like growth factor.     

Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.     

RAS蛋白在有丝分裂信号转导中的作用

致癌基因ｒａｓ编码的ＲＡＳ蛋白参与了多种细胞因子调控的有丝分裂过程。抗ＲＡＳ抗体能抑制细胞正常的有丝分裂。ｒａｓ基因转化的成纤维细胞ＮＩＨ３Ｔ３还具有在无血清和生长因子刺激下自发完成细胞周期的能力。在这种细胞中有活性氧的生成,抗氧化剂能抑制该细胞的有丝分裂。ＲＡＳ蛋白在有丝分裂的信号转导中参与了多条途径,是信号转导的重要组织者     

EGF receptor is involved in WNT3a-mediated proliferation and motility of NIH3T3 cells via ERK pathway activation

WNT3a stimulates proliferation of NIH3T3 cells via activation of the extracellular signal-regulated kinase (ERK) pathway. The RAF-1-->MEK-->ERK cascade was immediately increased by WNT3a treatment, however, the upstream event triggering ERK pathway activation by WNT3a is not clear. WNT3a activated RAS and WNT3a-induced ERK activation was blocked by dominant-negative RAS, indicating that WNT3a might act upstream of RAS. WNT3a-induced ERK pathway activations were blocked by AG1478, the epidermal growth factor receptor (EGFR) inhibitor, and EGFR siRNA. The WNT3a-induced ERK pathway activation was not observed in fibroblasts retaining defective EGFR, but the WNT3a effect was restored by EGFR reconstitution. These results indicate involvement of EGFR in the WNT3a-induced ERK pathway activation. WNT3a-induced motility and cytoskeletal rearrangement as well as proliferation of NIH3T3 cells were blocked by AG1478 and EGFR siRNA or abolished in EGFR knock-out fibroblasts, indicating involvement of EGFR in those cellular processes. WNT3a-induced ERK pathway activation was not affected by Dickkoff-1 (DKK-1), although WNT3a-induced activations of the WNT/beta-catenin pathway and proliferation were reduced by DKK-1. EGFR is involved in WNT3a-induced proliferation via both routes dependent on and independent of the WNT/beta-catenin pathway. These results indicate that WNT3a stimulates proliferation and motility of NIH3T3 fibroblasts via EGFR-mediated ERK pathway activation.     

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.     

Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ inter- acted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.     

Adenylate cyclase activity of NIH 3T3 cells morphologically transformed by ras genes

The observed homology between G-proteins which regulate adenylate cyclase and ras proteins and the suggested role of ras in the regulation of adenylate cyclase in yeast prompted us to examine the regulation of adenylate cyclase in three cell lines: (i) NIH 3T3 cells, (ii) NIH 3T3 cells transformed by high levels of the normal rasH gene product and (iii) NIH 3T3 cells transformed by a mutated rasH gene product. We found that the regulation of adenylate cyclase by G-proteins is identical in the three cell lines, although the response of the transformed NIH 3T3 cells to agonists is strongly attenuated. Our data suggest that mammalian ras products do not interact directly with adenylate cyclase, although their increased expression may indirectly inhibit the interaction of adenylate cyclase stimulatory receptors with G-proteins.     

The antiproliferative effects of agmatine correlate with the rate of cellular proliferation

Polyamines are small cationic molecules required for cellular proliferation. Agmatine is a biogenic amine unique in its capacity to arrest proliferation in cell lines by depleting intracellular polyamine levels. We previously demonstrated that agmatine enters mammalian cells via the polyamine transport system. As polyamine transport is positively correlated with the rate of cellular proliferation, the current study examines the antiproliferative effects of agmatine on cells with varying proliferative kinetics. Herein, we evaluate agmatine transport, intracellular accumulation, and its effects on antizyme expression and cellular proliferation in nontransformed cell lines and their transformed variants. H-ras- and Src-transformed murine NIH/3T3 cells (Ras/3T3 and Src/3T3, respectively) that were exposed to exogenous agmatine exhibit increased uptake and intracellular accumulation relative to the parental NIH/3T3 cell line. Similar increases were obtained for human primary foreskin fibroblasts relative to a human fibrosarcoma cell line, HT1080. Agmatine increases expression of antizyme, a protein that inhibits polyamine biosynthesis and transport. Ras/3T3 and Src/3T3 cells demonstrated augmented increases in antizyme protein expression relative to NIH/3T3 in response to agmatine. All transformed cell lines were significantly more sensitive to the antiproliferative effects of agmatine than nontransformed lines. These effects were attenuated in the presence of exogenous polyamines or inhibitors of polyamine transport. In conclusion, the antiproliferative effects of agmatine preferentially target transformed cell lines due to the increased agmatine uptake exhibited by cells with short cycling times. polyamines; antizyme; ornithine decarboxylase; polyamine transport     

Image analysis of Feulgen-stained NIH/3T3 cells transformed with DNA of 4-nitroquinoline 1-oxide treated human breast epithelial cells.

The nuclear phenotypes of Feulgen-stained NIH/3T3 cells transformed with 4-nitroquinoline 1-oxide (4NQO) treated, human breast epithelial cell (HBEC) DNA were studied by scanning microspectrophotometry and image analysis and compared with data obtained for nontransformed cells and for NIH/3T3 cells under ras oncogene transfecting situations. The Feulgen-DNA content of the individual nuclei (NQ1, NQ2, and NQ3 phenotypes) of the transformed cells was found not to be deeply affected, although presence of chromatin structures resembling double minutes could be verified in part of the metaphases of the transformed cells. On the other hand, the chromatin supraorganization of these cells showed some changes involving increased (NQ2, NQ3) or decreased (NQ1) levels of condensation. The changes in chromatin packing states, however, were of small magnitude compared with those reported for NIH/3T3 cells transfected with a c-H-ras oncogene or an N-ras-containing MCF-7 cell DNA. It was assumed that the transformation of the NIH/3T3 cells is not always necessarily accompanied by high levels of chromatin condensation. The transformation of the NIH/3T3 cells induced by the 4NQO-treated HBEC DNA and particularly the changes in chromatin condensation in these transformed cells could not be attributed merely to a ras activation elicited by the carcinogen. It is suggested that a more complex transforming mechanism is involved, probably owing to the fact that a whole genomic DNA of the 4NQO-treated HBEC has been used for transfection.     

TEL/JAK2 tyrosine kinase inhibits DNA repair in the presence of amifostine

The TEL/JAK2 chromosomal translocation (t(9;12)(p24;p13)) is associated with T cell childhood acute lymphoblastic leukemia. The TEL/JAK2 fusion protein contains the JAK2 catalytic domain and the TEL-specific oligomerization domain. TEL-mediated oligomerization of the TEL/JAK2 proteins results in the constitutive activation of the tyrosine kinase activity. Leukemia cells expressing TEL/JAK2 tyrosine kinase become resistant to anti-neoplastic drugs. Amifostine is a pro-drug which can selectively protect normal tissues against the toxicity of anticancer drugs and radiation. We investigated the effects of amifostine on idarubicin-induced DNA damage and repair in murine pro-B lymphoid BaF3 cells and BaF3-TEL/JAK2-transformed cells using alkaline single cell gel electrophoresis (comet assay). Idarubicin induced DNA damage in both cell types but amifostine reduced its extent in control non-transformed BaF3 cells and enhanced it in TEL/JAK2-transformed cells. The transformed cells did not show measurable DNA repair after exposure to amifostine and idarubicin, but cells treated only with idarubicin were able to recover within a 60-min incubation. Because TEL/JAK2-transformed cells can be considered as model cells for certain human leukemias and lymphomas we anticipate an enhancement of idarubicin cytotoxicity by amifostine in these diseases. Moreover, TEL/JAK2 tyrosine kinase might be involved in cellular response to DNA damage. Amifostine could promote apoptosis or lower the threshold for apoptosis induction dependent on TEL/JAK2 activation.     

Transformation of NIH 3T3 cells with basic fibroblast growth factor or the hst/K-fgf oncogene causes downregulation of the fibroblast growth factor receptor: reversal of morphological transformation and restoration of receptor number by suramin

When NIH 3T3 cells were transfected with the cDNA for basic fibroblast growth factor (bFGF), most cells displayed a transformed phenotype. Acquisition of a transformed phenotype was correlated with the expression of high levels of bFGF (Quarto et al., 1989). Cells that had been transformed as a result of transfection with bFGF cDNA had a decreased capacity to bind 125I-bFGF to high affinity receptors. NIH 3T3 cells transfected with bFGF cDNA that expressed lower levels of bFGF were not transformed and had a normal number of bFGF receptors. NIH 3T3 cells transfected with the hst/Kfgf oncogene, which encodes a secreted molecule with 45% homology to bFGF, also displayed a transformed phenotype and decreased numbers of bFGF receptors. However, NIH 3T3 cells transfected with the H-ras oncogene were transformed but had a normal number of bFGF receptors. Thus, transformation by bFGF-like molecules resulted in downregulation of bFGF receptors. Receptor number was not affected by cell density for both parental NIH 3T3 cells and transformed cells. In the cells transfected with bFGF cDNA that were not transformed, the receptors could be downregulated in response to exogenous bFGF. Conditioned medium from transformed transfected cells contained sufficient quantities of bFGF to downregulate bFGF receptors on parental NIH 3T3 cells. Thus, the downregulation of bFGF receptors seemed related to the presence of bFGF in an extracytoplasmic compartment. Treatment of the transformed transfected NIH 3T3 cells with suramin, which blocks the interaction of bFGF with its receptor, reversed the morphological transformation and restored receptors almost to normal numbers. These results demonstrate that in these cells bFGF transforms cells by interacting with its receptor and that bFGF and hst/K-fgf may use the same receptor.     

In vitro mesenchymal-epithelial transition in NIH3T3 fibroblasts results in onset of low-dose radiation hypersensitivity coupled with attenuated connexin-43 response

Background

Mesenchymal-to-epithelial transition (MET) is associated with altered cell adhesion patterns. Independent studies showed that cellular adhesion regulates low-dose hyper-radiosensitivity (HRS), a phenomenon reported widely in tumour cells. Therefore, present study aimed to investigate whether MET and associated cellular adhesion alterations affect cellular radiosensitivity.

Expression of retrovirus-related, cytotoxic T lymphocyte- and transplantation-defined antigens in NIH/3T3 transfectants after a single passage in nude mice

Transfection of T24c-Ha-ras oncogene into NIH/3T3 fibroblasts resulted in the establishment of a transformed cell line (pT) that was tumorigenic when injected s.c. both into Swiss outbred nude mice and normal NIH inbred mice. The passage into nude mice, however, led to the development of a tumor variant (pT-nude) able to subsequently grow into sublethally x-irradiated but not into immunocompetent NIH mice. NIH mice immunized with this tumor variant developed a strong specific CTL response against the immunizing cell line, whereas the parental transformed pT cell line was not lysed. Clones were derived by limiting dilution from anti-pT-nude bulk population and were tested on a panel of transformed NIH/3T3 lines before and after their growth as tumor into nude mice. All of these lines were lysed by the Lyt-2+ CTL clones as a sole consequence of one in vivo passage into nude mice. The cross-reactive Ag were shown to be related to endogenous retroviral products as assessed by 1) immunoprecipitations of gp70, p15E, and p30 viral proteins in the nude variants but not in parental lines, and 2) by the ability of retroviruses from irradiated pT-nude cells to infect NIH/3T3 or pT lines making them susceptible to lysis by anti-pT-nude CTL clones. These results show that a single passage in nude mice can induce retrovirus-related, cell-surface Ag in transplanted neoplastic cells.     

Involvement of nucleophosmin/B23 in the cellular response to curcumin

Nucleophosmin (NPM/B23) is a nucleolar phosphoprotein involved in cellular response to many different stimuli. Herein, we studied the molecular mechanism of NPM/B23 induction by curcumin, a natural AP-1 inhibitor with antitumor properties. Exposure to 5-30 μM curcumin significantly and dose-dependently increased the level of NPM/B23 in non-transformed NIH 3T3 cells but not HeLa cells and F9 cells. Besides, the transformed F9 and HeLa cells are more sensitive to curcumin-induced cell death and growth inhibition than NIH 3T3 cells. Overexpression of c-Jun, but not c-Fos, decreased ∼40% of NPM/B23 and enhanced the sensitivity of NIH 3T3 cells to 30 μM curcumin. Furthermore, down-regulation of NPM/B23 by transfection with NPM/B23 antisense plasmid enhanced the sensitivity to curcumin-induced cell death and growth inhibition. These results indicated that NPM/B23 expression regulates cellular sensitivity to curcumin. Besides, NPM/B23 knockdown may facilitate as a novel strategy to promote the sensitivity of cancer cells to curcumin.     

Structure of the provirus within NIH 3T3 cells transfected with Harvey sarcoma virus DNA.

NIH 3T3 cells transformed with unintegrated Harvey sarcoma virus (HSV) linear DNA generally acquired a complete HSV provirus. Infection of these transformed cells with Moloney murine leukemia helper virus was followed by release of infectious particles. The HSV provirus within these transfected cells was convalently joined to nonviral DNA sequences and was termed "cell-linked" HSV DNA. The association of this cell-virus DNA sequence with the chromosomal DNA of a transfected cell was unclear. NIH 3T3 cells could also become transformed by transfection with this cell-linked HSV DNA. In this case, the recipient cells generally acquired a donor DNA fragment containing both the HSV provirus and its flanking nonviral sequences. After cells acquired either unintegrated or cell-linked HSV DNA, the newly established provirus and flanking cellular sequences underwent amplifications to between 5 and 100 copies per diploid cell. NIH 3T3 cells transfected with HSV DNA may acquire deleted proviral DNA lacking at least 1.3 kilobase pairs from the right end of full-length HSV 6-kilobase-pair DNA (corresponding to the 3'-proximal portion of wild-type HSV RNA). Cells bearing such deleted HSV genomes were transformed, indicating that the viral transformation gene lies in the middle or 5'-proximal portion of the HSV RNA genome. However, when these cells were infected with Moloney murine leukemia helper virus, only low levels of biologically active sarcoma virus particles were released. Therefore, the 3' end of full-length HSV RNA was required for efficient transmission of the viral genome.