The sensitivity to growth factors of NIH 3T3 cells transformed by the v-myc oncogene

Transformation of NIH 3T3 cells, induced by v-myc oncogene, activates a proliferative potential of the cells cultivated in the serum-free medium, and reduces the ratio of 3H-Tdr incorporation into the cells grown in the presence of 10% fetal serum in comparison to those grown in the serum-free medium. The v-myc transformed cells (NIH 3T3-v-myc) as well as the untransformed ones are very responsive to insulin. On the other hand, the epidermal growth factor, able to stimulate proliferation of NIH 3T3 cells, exert no effects on the NIH 3T3-v-myc cells. The NIH 3T3-v-myc cells cultivated in the medium, containing 2.5% human plasma enriched with thrombocytes, have the same proliferative characteristics as cells grown in the thrombocyte-free plasma. It is concluded that transformation of NIH 3T3 cells induced by v-myc oncogene may reduce a requirement for thrombocyte-released growth factors and EGF but not for insulin.     

Abelson murine leukemia virus induces platelet-derived growth factor-independent fibroblast growth: correlation with kinase activity and dissociation from full morphologic transformation.

Abelson murine leukemia virus (A-MuLV) encodes a single protein product, a tyrosine-specific protein kinase, whose activity is necessary for cell transformation by this retrovirus. Using a defined medium culture system, we demonstrate that transformation of NIH 3T3 fibroblasts by A-MuLV abrogates their normal requirement for platelet-derived growth factor (PDGF) for cell growth. Analysis of constructed insertional mutant viruses revealed an absolute correlation between A-MuLV-encoded tyrosine kinase activity and PDGF-independent fibroblast growth. Sequences of the provirus not required for kinase activity appeared unnecessary for abrogating the fibroblast requirement for PDGF. Conversely, sequences required for kinase activity appeared necessary, suggesting that induction of PDGF-independent fibroblast growth, like cell transformation, is a function of this tyrosine kinase. Fibroblasts transformed by a partially transformation-defective mutant demonstrated incomplete morphological transformation but were still independent of PDGF for growth. Thus, the processes of full morphological transformation and growth factor independence can be partially dissociated.     

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.     

Analysis of the reduced growth factor dependency of simian virus 40-transformed 3T3 cells.

We have measured in a defined serum-free medium the platelet-derived growth factor (PDGF) and insulin requirements of normal Swiss 3T3 cells, simian virus 40-transformed 3T3 cells, and partial revertants of simian virus 40-transformed 3T3 cells. Swiss 3T3 cells displayed strong requirements for both PDGF and insulin. Both of these requirements were significantly diminished in simian virus 40-transformed 3T3 cells. Analysis of the PDGF and insulin requirements of the revertants indicated that the loss of either of these two growth factor requirements was not necessarily linked to the other; rather, the growth factor requirements were specifically associated with other parameters of transformation. The reacquisition of a PDGF requirement cosegregated with reversion to density-dependent growth inhibition, whereas reacquisition of a normal insulin requirement cosegregated with reversion to a normal growth dependence on calf serum. Anchorage dependence was dissociable from both growth factor requirements. The relationship between the PDGF requirement and density-dependent growth inhibition was further analyzed in normal 3T3 cells by measuring the PDGF requirement at different cell densities. At high cell densities, the requirement for PDGF became significantly greater. We suggest that at least in part the ability of transformed cells to grow to high saturation densities results from their loss of a requirement for PDGF.     

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.     

Tumorigenic transformation of NIH 3T3 cells by the autocrine synthesis of transforming growth factor alpha

A variety of cancer cells overexpress transforming growth factor alpha (TGF alpha), a mitogenic peptide. A cDNA sequence coding for the full-length human TGF alpha precursor protein was subcloned into a retroviral expression vector and introduced into clone 7 NIH 3T3 cells, which have low numbers of endogenous epidermal growth factor receptors (EGFRs). The autocrine synthesis of TGF alpha by these cells resulted in their focal transformation. In contrast, control NIH 3T3 cells treated in a paracrine manner with exogenous, saturating concentrations of the mature form of TGF alpha, though stimulated to divide, remained morphologically untransformed. The addition of saturating quantities of soluble, mature TGF alpha to NIH 3T3 cells expressing the transferred TGF alpha gene actually suppressed their growth and focal transformation. The transformation induced by the TGF alpha gene remained an EGFR-dependent process, since the degree of transformation was correlated with EGFR expression in NIH 3T3 cells and since NR6 cells, which are Swiss 3T3 cells devoid of endogenous EGFRs, were transformed by the TGF alpha vector only when exogenous EGFR genes were also introduced. When inoculated into nude mice, the TGF alpha-expressing cells rapidly gave rise to tumors that grew progressively, whereas control cells did not form tumors. We conclude that in certain circumstances autocrine TGF alpha can be more oncogenic than paracrine and that paracrine TGF alpha can suppress this effect.     

EphA3基因稳定表达细胞模型的建立与分析

目的：建立稳定表达外源EphA3基因的小鼠成纤维细胞株模型,初步探讨EphA3基因表达对肿瘤发生、发展的影响。方法：通过脂质体介导的方法,将真核表达载体pcDNA3.1（-）/myc-his-EphA3转染NIH3T3细胞,用Western印迹确定外源EphA3基因表达;通过MTT实验、软琼脂集落形成实验,观察EphA3基因表达对NIH3T3细胞生物学特性的影响。结果：建立了稳定转染EphA3基因的NIH3T3细胞株;EphA3基因表达的小鼠成纤维NIH3T3细胞生长速度没有明显变化,但在软琼脂上锚着非依赖生长的能力加强。结论：建立了稳定表达外源EphA3基因的NIH3T3细胞株,EphA3基因稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。     

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.     

FGF3 attached to a phosholipid membrane anchor gains a high transforming capacity. Implications of microdomains for FGF3 cell transformation

NIH3T3 cells transformed by mouse FGF3-cDNA (DMI cells) selected for their ability to grow as anchorage-independent colonies in soft agar and in defined medium lacking growth factors exhibit a highly transformed phenotype. We have used dominant negative (DN) fibroblast growth factor (FGF) receptor 2 (FGFR2) isoforms to block the FGF response in DMI cells. When the DN-FGFR was expressed in DMI cells, their transformed phenotype can be reverted. The truncated FGFR2(IIIb), the high affinity FGFR for FGF3, is significantly more efficient at reverting the transformed phenotype as the IIIc isoform, reaffirming the notion that the affinity of the ligand to the DN-FGFR2 isoform determines the effect. Heparin or heparan sulfate displaces FGF3 from binding sites on the cell surface inhibiting the growth of DMI cells and reverts the transformed phenotype (). However, the presence of heparin is necessary to induce a mitogenic response in NIH3T3 cells when stimulated with soluble purified mouse FGF3. We have investigated the importance of cell surface binding of FGF3 for its ability to transform NIH3T3 cells by creating an FGF3 mutant anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI anchor renders the cell surface association of FGF3 independent from binding to heparan sulfate-proteoglycan of the cell surface membrane. Attachment of a GPI anchor to FGF3 also confers a much higher transforming potential to the growth factor. Even more, the purified GPI-attached FGF3 is as much transforming as the secreted protein acting in an autocrine mode. Because NIH3T3 cells do not express the high affinity tyrosine kinase FGF receptors for FGF3, these findings suggest that FGF3 attached to GPI-linked heparan sulfate-proteoglycan may have a broader biological activity as when bound to transmembrane or soluble heparan sulfate-proteoglycan.     

Amplification and overexpression of the met gene in spontaneously transformed NIH3T3 mouse fibroblasts.

We have identified a class of transformed NIH3T3 mouse fibroblasts that arise at low frequencies in transfection experiments with DNA from both neoplastic and non-neoplastic cells and that may result from a low level of spontaneous transformation of NIH3T3 cells. DNA from the transformed cells was unable to transform NIH3T3 cells in a second cycle of transfection and, where examined, the cells showed no evidence for the uptake of the transfected DNA sequences. The results of Southern analyses demonstrate that a mouse homologue of the human met oncogene is amplified 4- to 8-fold in 7 of 10 lines of these transformed NIH3T3 mouse fibroblasts. The cells containing the amplified gene also exhibit at least a 20-fold overexpression of an 8.5-kb mRNA that is homologous to met. To test the hypothesis that met encodes a growth factor receptor, we examined the binding of platelet-derived growth factor, epidermal growth factor, insulin-like growth factor I and gastrin-releasing peptide to transformed and non-transformed NIH3T3 cells. The results show that there is no significant elevation of the binding of these growth factors to cells containing amplification and overexpression of met.     

Cell transformation as aberrant differentiation：Environmentally dependent spontaneous transformation of NIH 3T3 cells

NIH 3T3 cells, a mouse fibroblast cell line used as routine target cells for transfection experiments, undergo spontaneous transformation in our experiments after they form a confluent sheet in medium containing fetal bovine serum (FBS) or lower coneentration of calf serum (CS). The transformation takes the form of foci of multiplying cells among the surrounding cells which have stopped cell division. However, no focus of transformed cells could be seen in medium containing high concentration (10％) of CS. Further experiments indicated that the frequency of transformation is highly dependent on the concentration of serum and the transformation in CS is changeable when the cells are passaged in FBS. 3~H-thymidine autoradiography has been proved to be a sensitive measurement indicator for focus formation. Our results suggest that the high frequency of transformation and its dependence on confluency as well as on medium composition are characteristics of cell differentiation rather than mutation. The role of the NIH 3T3 cell line as a cancer-initiated cell population and its accelerated transformation by ras oncogene might be considered as a form of tumor promotion is discussed.     

Cooperation of middle and small T antigens of polyomavirus in transformation of established fibroblast and epithelial-like cell lines.

We have reported recently that small T antigen of polyomavirus stimulates the growth of NIH 3T3 cells beyond their saturation density and induces weak anchorage-independent growth (T. Noda, M. Satake, T. Robins, and Y. Ito, J. Virol. 60:105-113, 1986). We examined whether small T antigen would cooperate with middle T antigen in the in vitro transformation of NIH 3T3 (fibroblasts) and NRK-52E (epitheliallike) cells. The small-T-antigen gene, when cotransfected with the middle-T-antigen gene, had no additional effect on the efficiency or size of dense foci formation induced by the middle-T-antigen gene on a monolayer of NIH 3T3 cells. However, the small-T-antigen gene dramatically increased the rate of growth of NIH 3T3 cells transformed by middle T antigen in semisolid medium. Introduction of the small-T-antigen gene into middle-T-antigen-transformed cells did not disturb the integrated middle-T gene, alter expression of the middle-T gene, or enhance middle-T-antigen-associated tyrosine protein kinase activity. For NRK-52E cells, the expression of middle T antigen alone resulted in small, slow-growing foci on a monolayer. These cells did not show anchorage-independent growth, despite the fact that middle-T-antigen-associated tyrosine protein kinase activity was clearly detected in these cells. NRK-52E cells expressing both middle and small T antigens formed faster growing foci on a monolayer than middle-T-antigen-expressing cells did and grew in semisolid medium, even when the amounts of middle T antigen and its associated kinase activities were lower than those of middle-T-antigen-expressing cells. We conclude that small T antigen cooperates with middle T antigen in the in vitro transformation of established cell lines of fibroblast and epitheliallike cells, that it does not share the middle-T-antigen function even though they are structurally related, and that it has a significantly more important role in the transformation of NRK-52E cells than that of NIH 3T3 cells.     

V-fos基因对NIH3T3细胞转化的研究

本文报道了用含v-fos基因的pFBJ-2质粒转染NIH 3 T 3细胞,获得了转化细胞株。对细胞株的研究结果表明:(1)Southern杂交检测到细胞基因组中有v-fos基因的整合;(2)点杂交测得有v-fos mRNA的表达;(3)出现一系列转化表型,包括细胞形态的改变,异常的增长速率,在软琼脂上的贴壁不依赖性生长,对低血清浓度培养液的适应性以及细胞膜表而超微结构的变化等,提示v-fos基因能使NIH 3 T 3细胞发生转化,并在体外转化过程中起决定性作用。     

Regulation of transformed state by calpastatin via PKCepsilon in NIH3T3 mouse fibroblasts.

Ca(2+)-activated neutral protease calpain is ubiquitously expressed and may have pleiotropic biological functions. We have previously reported that repeated treatment of NIH3T3 mouse fibroblasts with the calpain inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) resulted in the induction of transformed foci [T. Hiwasa, T. Sawada, and S. Sakiyama (1990) Carcinogenesis 11, 75-80]. To elucidate further the effects of calpain in malignant transformation of NIH3T3 cells, calpastatin, an endogenous specific inhibitor of calpain, was expressed in NIH3T3 cells by transfection with cDNA. G418-selected calpastatin-expressing clones showed a significant increase in the anchorage-independent growth ability. A similar increase in cloning efficiency in soft agar medium was also observed in calpain small-subunit-transfected clones. On the other hand, reduced expression of calpastatin achieved by transfection with calpastatin antisense cDNA in Ha-ras-transformed NIH3T3 (ras-NIH) cells caused morphological reversion as well as a decrease in anchorage-independent growth. When NIH3T3 cells were treated with ALLN for 3 days, cell growth was stimulated by approximately 10%. This growth stimulation by ALLN was not observed in ras-NIH cells, but recovered by expression of a dominant negative form of protein kinase C (PKC)epsilon but not by that of PKCalpha. Western blotting analysis showed that an increase in PKCepsilon was much more prominent than that of PKCalpha in NIH3T3 cells after treatment with ALLN. These results are concordant with the notion that calpain suppresses malignant transformation by predominant degradation of PKCepsilon.     

Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA.

The expression of human basic fibroblast growth factor (bFGF) cDNA in mouse BALB/c 3T3 clone A31 cells induced morphological transformation. These transformed cells grew well and reached more than a sixfold-higher saturation density than parental A31 cells even in serum-free medium. They were able to form colonies in soft agar. The phenotypic alteration in the transformed cells was reversed by the addition of anti-human bFGF antibodies to the medium. These results suggest that the cellular transformation mediated by bFGF is caused by autocrine stimulation with secreted bFGF molecules.     

Expression of protein kinase C I in NIH 3T3 cells increases its growth response to specific activators

In order to investigate the effects of protein kinase C (PKC) expression on cellular growth and morphology, we established mouse fibroblast cell populations which expressed the rat pkc-γ gene under the control of a retroviral promoter. NIH 3T3 stable transfectants displayed a three-fold increase in total PKC levels. These cells appeared morphologically unaltered but exhibited a stronger mitogenic response to 12-O-tetradecanoylphorbol-13-acetate (TPA) and cardiolipin (CL) as well as enhanced growth in semisolid medium in the presence of TPA. Thus, at these enzyme levels, PKC conferred growth advantages to NIH 3T3 cells only in response to specific activators.     

Transformed phenotype conferred to NIH/3T3 cells by ectopic expression of heparin-binding growth factor 1/acidic fibroblast growth factor

Summary Heparin-binding growth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen and angiogenic factor found in tissues such as brain, kidney and heart. The genomic and cDNA sequences indicate that HBGF-1 does not have a typical signal peptide sequence. HBGF-1 was shown to be localized to the extracellular matrix of cardiac myocytes, but the mechanism of secretion is not presently known. We have cloned the HBGF-1 cDNA which allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. A previous report showed that the truncated HBGF-1 confers partial transformed phenotype to the recipient fibroblasts. However, expression of full-length HBGF-1 has not been reported. The HBGF-1 coding sequence was cloned into the retroviral expression vector, SVX, and transfected into NIH/3T3 cells. Transfectants expressing full-length HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 is a unique band of approximately 20 kDa and can be detected in the cell homogenate but not in the conditioned medium. NIH/3T3 cells were conferred anchorage independence when HBGF-1 was provided exogenously. We showed the transformed cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfected cells expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus, NIH/3T3 cells acquired a full spectrum of transformed phenotype when full length HBGF-1 was expressed at high levels. This work was supported by grants from the National Cancer Institute (RO1 CA45611), The March of Dimes Birth Defects Foundation (No. 6-549) and The Ohio Cancer Research Associates, Inc. I.-M.C. is a recipient of The Research Career Development Award (KO4 CA01369) from the National Institutes of Health.     

Modulation of cell growth and transformation by doxycycline-regulated FGF-2 expression in NIH-3T3 cells.

The single-copy fibroblast growth factor 2 (FGF-2) gene encodes four coexpressed isoforms of different molecular masses. The 18-kDa FGF-2 is primarily localized in the cytoplasm, whereas the higher molecular mass isoforms (HMW FGF-2) localize to the nucleus and nucleolus. The overexpression of either 18-kDa FGF-2 or HMW FGF-2 promotes cell transformation in a dose-dependent manner. In NIH 3T3 cells, the selective overexpression of HMW FGF-2 but not of 18-kDa FGF-2 confers upon the cells the unique phenotype of growth in low serum-containing medium. Thus, the distinct intracellular localization and the level of expression of FGF-2 are pivotal requirements for the differential effects of FGF-2 isoforms on the cellular phenotype. On this basis, we established a doxycycline-regulatable FGF-2 expression system that permitted us to regulate the expression of each isoform in a time- and dose-dependent manner. We analyzed the growth properties of cells in the presence and absence of doxycycline in both normal and low serum-containing medium and in soft agar. The doxycycline-activated expression of 18-kDa FGF-2 did not allow growth in low serum medium. The growth of cells expressing HMW FGF-2 was increased by doxycycline under all three conditions, and a relationship between the level of HMW FGF-2 expression and cell growth was observed for all three conditions. This doxycycline-regulatable FGF-2 expression system provides a mechanism to analyze changes in FGF-2 targeted pathways and genes and to characterize pathways specifically activated by either the 18-kDa FGF-2 or the HMW FGF-2 isoforms.     

过表达Gankyrin基因细胞模型的建立及其成瘤性分析

目的：Gankyrin作为一个新的癌基因,在细胞周期调控和肿瘤发生过程中有重要功能。建立Gankyrin过表达的细胞和动物模型,以便进一步研究其在肿瘤形成过程中的作用机制。方法：采用逆转录病毒感染细胞的方法构建Gankyrin稳定表达的细胞株,采用Western-blot检测Gankyrin的表达,采用软琼脂和裸鼠成瘤实验验证该细胞株的恶性转化效应。结果：构建了Gankyrin稳定过表达的NIH3T3细胞株,且该细胞株具有成瘤性。结论：采用逆转录病毒感染细胞的方法可以有效建立Gankyrin转化细胞株,为进一步研究Gankyrin的作用机制及其在肿瘤形成中的功能提供了基础。