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。     

High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing the lowest PiT1 mRNA levels. The mechanism behind the role of PiT1 in increased cell proliferation is not known. We, however, found that compared to control cells, cultures of NIH3T3 cells overexpressing PiT1 upon seeding showed increased cell number after 24 h and had shifted more cells from G0/G1 to S+G2/M within 12 h, suggesting that an early event may play a role. We here show that expression of human PiT1 in NIH3T3 cells led to faster cell adhesion; this effect was not cell type specific in that it was also observed when expressing human PiT1 in MC3T3-E1 cells. We also show for NIH3T3 that PiT1 overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby contribute to their proliferative advantage within the first 24 h after seeding.     

Properties of a murine retroviral recombinant of avian acute leukemia virus E26: a murine fibroblast assay for v-ets function.

A replication-defective murine retroviral construct, termed pME26, was generated by inserting avian gag-myb-ets sequences derived from the cloned avian acute leukemia virus E26 into an Abelson murine leukemia virus-derived retroviral vector. ME26 virus can be rescued efficiently from transfected NIH 3T3 cells by replicating murine leukemia viruses. Either pME26-transfected nonproducers or ME26 virus-infected NIH 3T3 cells expressed a 135-kilodalton fusion protein (p135) which was detectable by immunoprecipitation with antiserum directed against avian leukemia virus p27gag, myb or ets oncogene protein, or murine leukemia virus p15gag and was principally localized in the nucleus. NIH 3T3 cells infected with ME26 exhibited morphological alterations and increased proliferation in reduced serum and formed small colonies in agar suspension. Discrete foci could be readily recognized in cells maintained in a defined medium containing 0.03 to 0.1% calf serum. In newborn NFS/N mice, ME26 induced a significantly higher mortality and incidence of erythroid and myeloid leukemias. Analysis of a series of mutants affecting the expression of various portions of p135 indicated that the v-ets gene acts to mitogenically stimulate the proliferation of NIH 3T3 fibroblasts and reduces or abolishes their serum dependence. These properties provide an assay system to study functions of the ets gene family.     

Temperature-sensitive mutants of Abelson murine leukemia virus deficient in protein tyrosine kinase activity.

The effect of two missense mutations in abl on transformation by Abelson murine leukemia virus was evaluated. These mutations led to the substitution of a histidine for Tyr-590 and a glycine for Lys-536. Both changes gave rise to strains that were temperature dependent for transformation of both NIH 3T3 cells and lymphoid cells when expressed in the context of a truncated Abelson protein. In the context of the prototype P120 v-abl protein, the Gly-536 substitution generated a host range mutant that induced conditional transformation in lymphoid cells but had only a subtle effect on NIH 3T3 cells. The combination of both substitutions gave rise to a P120 strain that was temperature sensitive for both NIH 3T3 and lymphoid cell transformation. The Abelson proteins encoded by the temperature-sensitive strain displayed in vitro kinase activities that were reduced when compared with those of wild-type proteins. In vivo, levels of phosphotyrosine were reduced only at the restrictive temperature. Analysis of cells expressing either the wild-type P160 v-abl protein or the P210 bcr/abl protein and an Abelson protein encoded by a temperature-sensitive strain failed to correct this defect, suggesting either that tyrosine phosphorylation in vivo is an intramolecular reaction or that the protein encoded by the temperature-sensitive strain is a poor substrate for tyrosine phosphorylation in vivo. These results raise the possibility that tyrosine phosphorylation of Abelson protein plays a role in transformation.     

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals¹. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)². When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci¹ that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.     

Phosphorylation of acyclovir [9-(2-hydroxyethoxymethyl)guanine] in Epstein-Barr virus-infected lymphoblastoid cell lines.

The extent of phosphorylation of 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV]) in fresh peripheral leukocytes, in Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines, and in herpes simplex virus type 1-infected lymphoblastoid (P3HR-1) and monkey kidney (Vero) cells was determined by high-pressure liquid chromatography, Mono-, di-, and triphosphorylated derivatives of [8-14C]ACV were detected at low levels at various times after superinfection of Raji cells with EBV. The extent of phosphorylation appeared to be related to the concentration of ACV in the medium. Small amounts of ACV mono-, di-, and triphosphates were formed in fresh peripheral leukocyte preparations from EBV- seropositive and -seronegative donors. Comparable ACV monophosphate levels were detected in EBV-negative BJAB and the EBV-positive BJAB/GC cell lines; however, no di- or triphosphate derivatives were detected. Comparable ACV-monophosphate levels were detected in both P3HR-1 and HSV-infected P3HR-1 cell lines; however, larger amounts of ACV di- and triphosphorylated derivatives were detected in the HSV-infected P3HR-1 cells. ACV was converted to the triphosphate to a greater extent in HSV-infected Vero cells than in mock-infected Vero cells or in HSV-infected P3HR-1 cells. ACV or its phosphorylated derivatives were converted to guanine nucleotides to a greater extent in lymphoblastoid cells than in fibroblasts (Vero). In conclusion, neither the productive replication of EBV nor the presence of latent viral DNA is required for ACV monophosphate formation in B lymphoblastoid cells. ACV triphosphate, however, was detected only in cells infected productively with EBV.     

Induction of Syncytia by Neuropathogenic Murine Leukemia Viruses Depends on Receptor Density, Host Cell Determinants, and the Intrinsic Fusion Potential of Envelope Protein

Infection by the neuropathogenic murine leukemia virus (MLV) TR1.3 results in hemorrhagic disease that correlates directly to in vivo syncytium formation of brain capillary endothelial cells (BCEC). This phenotype maps to amino acid 102 in the envelope (Env) protein of TR1.3. Substitution of glycine (G) for tryptophan (W) at this position (W102G Env) in the nonpathogenic MLV FB29 induces both syncytium formation and neurologic disease in vivo. Using an in vitro gene reporter cell fusion assay, we showed that fusion either with murine NIH 3T3 cells or with nonmurine target cells that expressed receptors at or below endogenous murine levels mirrored that seen in BCEC in vivo. In these instances only TR1.3 and W102G Env induced cell fusion. In contrast, when receptor levels on nonmurine cells were raised above endogenous murine levels, FB29 Env was as fusogenic as the neuropathogenic TR1.3 and W102G Env. These results indicate that TR1.3 Env and W102G Env are intrinsically more fusogenic than FB29 Env, that the induction of fusion requires a threshold number of receptors that is greater for FB29 Env than for TR1.3 or W102G Env, and that receptor density on murine NIH 3T3 cells and BCEC is below the threshold for FB29-dependent fusion. Surprisingly, receptor density on NIH 3T3 cells could not be increased by stable expression of exogenous receptors, and FB29-dependent fusion was not observed in NIH 3T3 cells that transiently expressed elevated receptor numbers. These results suggest that an additional undefined host cell factor(s) may limit both receptor expression and fusion potential in murine cells.     

脑源性神经营养因子受体trkB在NIH 3T3细胞上的表达

构建了克隆有大鼠脑源性神经营养因子（BDNF）受体trkB全长基因的真核表达载体pcDNA3.1( )-rat trkB.用脂质体介导法将重组载体转入小鼠NIH3T3细胞,在mRNA和蛋白质水平检测到了trkB基因在用G418筛选到的抗性NIH 3T3细胞中的表达,表达的trkB蛋白定位于细胞膜上。BDNF能够剂量依赖性地促进NIH 3T3－trkB细胞的增殖,说明表达的trkB是有功能的。该表达trkB和NIH3T3细胞为研究BDNF的生理功能、活性测定和从噬菌体展示肽库中筛选BDNF肽提供了一个简便的细胞模型。     

Activation of the Murine Sarcoma Virus Genome After Infection with the Murine Leukemia Virus as Determined by Cell Agglutination

Non-virus-producing NIH/3T3 cells transformed by the murine sarcoma virus are agglutinated by conconavalin A to the same low level as normal NIH/3T3 cells. Infection with the murine leukemia virus greatly increases the agglutination of transformed cells but not that of normal cells. These data suggest that the morphological expression of cell transformation and the surface alterations associated with increased cell agglutination are controlled by the expressions of different sarcoma virus genes.     

Tumorigenicity of the met proto-oncogene and the gene for hepatocyte growth factor.

The met proto-oncogene is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). It was previously shown that, like the oncogenic tpr-met, the mouse met proto-oncogene transforms NIH 3T3 cells. We have established NIH 3T3 cells stably expressing both human (Methu) and mouse (Metmu) met proto-oncogene products. The protein products are properly processed and appear on the cell surface. NIH 3T3 cells express endogenous mouse HGF/SF mRNA, suggesting an autocrine activation mechanism for transformation by Metmu. However, the tumor-forming activity of Methu in NIH 3T3 cells is very low compared with that of Metmu, but efficient tumorigenesis occurs when Methu and HGF/SFhu are coexpressed. These results are consistent with an autocrine transformation mechanism and suggest further that the endogenous murine factor inefficiently activates the tumorigenic potential of Methu. The tumorigenicity observed with reciprocal chimeric human and mouse receptors that exchange external ligand-binding domains supports this conclusion. We also show that HGF/SFhu expressed in NIH 3T3 cells produces tumors in nude mice.     

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.     

Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1

We constructed lambda recombinants containing the Harvey murine sarcoma virus genome and the thymidine kinase (tk) gene of herpes simplex virus type 1 linked to each other. The tk gene was located in a position downstream from both the long terminal repeat and the src gene of Harvey murine sarcoma virus. The DNAs of the lambda recombinants were used to transfect NIH3T3 mouse fibroblasts in order to obtain Harvey murine sarcoma virus DNA-induced foci of transformed cells. The transformed foci were superinfected with a helper-independent retrovirus, and new individual retrovirus were isolated from the superinfected foci. The new viruses could induce focus formation on NIH3T3 cells and could convert NIH3T3(TK-) cells into TK+ cells by carrying the herpes simplex virus type 1 tk gene into the TK- cells. From virus-infected cells, we isolated nonproducer foci on NIH3T3 cells and TK+ transformants on NIH3T3(TK-) cells containing one such new viral genome coding for the dual properties. The new retroviral sequence in the nonproducer cells could be rescued into virus particles at high titers by superinfection with a helper-independent retrovirus. A hybridization analysis indicated that the recombinant virus contained both the Harvey murine sarcoma virus src sequence and the tk gene sequence in a single RNA species approximately 4.9 kilobases long. We concluded that retroviruses can be used as true vectors for genes other than genes that lead to oncogenesis.     

Site-selective cAMP analogs induce nuclear translocation of the RII cAMP receptor protein in Ha-MuSV-transformed NIH/3T3 cells

Site-selective cAMP analogs, depending on the position of their substituents on the adenine ring, selectively bind to either site 1 or site 2 of the known cAMP binding sites of protein kinase. Treatment of Harvey murine sarcoma virus-transformed NIH/3T3 cells with such site-selective analogs results in growth inhibition and phenotypic reversion, and the combination of a C-8 thio or halogen analog (site 1 selective) with an N6 analog (site 2 selective) produces a synergistic effect. We report here that the growth inhibitory effect of the analogs correlates with the nuclear translocation of the RII cAMP receptor protein, the regulatory subunit of protein kinase type II. The transformed NIH/3T3 cells contained no detectable level of RII in the nucleus, whereas nontransformed NIH/3T3 cells exhibited a high level of nuclear RII. Within 30 min after treatment of the transformed cells with the site-selective analogs, immunofluorescence against the RII protein markedly increased in the cell nucleus. The nuclear translocation of the RII cAMP receptor protein is an early event in the reverse transformation of the fibroblasts treated with site-selective cAMP analogs.     

Murine Sarcoma and Leukemia Viruses: Assay Using Clonal Lines of Contact-Inhibited Mouse Cells

A continuous cell line of highly contact-inhibited cells (NIH/3T3) has been developed from NIH Swiss mouse embryo cultures. Its growth properties are similar to those of 3T3 and BALB/3T3. Although 3T3 is relatively insensitive to focus formation by murine sarcoma viruses, cloned lines of both NIH/3T3 and BALB/3T3 have been isolated that are highly sensitive to sarcoma virus focus formation and leukemia virus growth. The sensitivity and specificity are comparable to those found with primary embryo cells. MSV-transformed lines of NIH/3T3 have been obtained.     

Cell fusion induced by the murine leukemia virus envelope glycoprotein.

To determine whether ecotropic murine leukemia virus (MuLV) envelope glycoproteins are sufficient to cause cell-to-cell fusion when expressed in the absence of virus production, we used an ecotropic MuLV, AKV, to construct env expression vectors that lack the gag and pol genes. The rat cell line XC, which undergoes cell-to-cell fusion upon infection with ecotropic MuLV, was transfected with wild-type env expression vectors, and high levels of syncytium formation resulted. Transfection of the murine cell line NIH 3T3 with expression vectors containing the wild-type or mutated env region did not result in syncytium formation. Immunoprecipitation analysis of the envelope glycoproteins expressed in NIH 3T3 and XC cells showed that the mature surface glycoprotein expressed in XC cells was of a much lower apparent molecular weight than that expressed in NIH 3T3 cells. Further characterization showed that most if not all of this difference was the result of differences in glycosylation. Finally, site-directed mutagenesis was used to introduce several conservative and nonconservative changes into the amino-terminal region of the transmembrane protein. Analysis of the effect of these mutations confirmed that this region is a fusion domain.     

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.