Transfection of EK-3, a subline of NIH 3T3, with the oncogene Ha-ras does not abolish its anchorage dependence

EK-3 cells, previously isolated by us from cultures of NIH 3T3, require both ras and myc oncogenes for efficient transformation, while their parent cells are readily transformed by ras alone. We transfected the EK-3 cells with the v-Ha-ras oncogene and obtained several sublines which integrated this gene and transcribed it successfully. The ras-NIH 3T3 formed foci of multilayered cells that were piling up in culture, while the ras-EK-3 cells remained contact inhibited. Furthermore, when the growth of the cells in soft agar was examined, a clear difference was observed. Cells of the ras-NIH 3T3 clonal lines showed high efficiency of growth (10%), while the ras-EK-3 cells exhibited low efficiency (0.2%). The latter being quite similar to that of the non-transfected NIH 3T3 and EK-3 cells (0.05%). The results presented now, showing that ras-EK-3 cells are more anchorage dependent than the ras-NIH 3T3 cells, clearly indicate that differences, previously shown to exist between EK-3 and NIH 3T3 cells, persist in their daughter cell lines derived following transfection with the Ha-ras oncogene.     

Mitotic clonal expansion during preadipocyte differentiation: calpain-mediated turnover of p27

Evidence is presented that calpain, a calcium-activated protease, degrades the cyclin-dependent kinase inhibitor, p27, during the mitotic clonal expansion phase of 3T3-L1 preadipocyte differentiation. Calpain activity is required during an early stage of the adipocyte differentiation program. Thus, inhibition of calpain with N-acetyl-Leu-Leu-norleucinal (ALLN) blocks clonal expansion and acquisition of the adipocyte phenotype only when added between 12 and 24 h after the induction of differentiation. Likewise, inhibition of calpain by overexpression of calpastatin, the specific endogenous inhibitor of calpain, prevents 2-day post-confluent preadipocytes from reentering the cell cycle triggered by the differentiation inducers. Inhibition of calpain with ALLN causes preadipocytes to arrest just prior to S phase and prevents phosphorylation of the retinoblastoma gene product, DNA replication, clonal expansion, and subsequent adipocyte differentiation but does not affect the expression of immediate early genes (i.e. fos, jun, C/EBPbeta, and C/EBPdelta). Inhibition of calpain by either ALLN or by overexpression of calpastatin blocks the degradation of p27. p27 is degraded in vitro by cell-free extracts from clonally expanding preadipocytes that contain "active" calpain but not by extracts from pre-mitotic preadipocytes that do not. This action is inhibited by calpastatin or ALLN. Likewise, p27 in preadipocyte extracts is a substrate for purified calpain; this proteolytic action was inhibited by heat inactivation, EGTA, or ALLN. Thus, extracellular signals from the differentiation inducers appear to activate calpain, which degrades p27 allowing density-dependent inhibited preadipocytes to reenter the cell cycle and undergo mitotic clonal expansion.     

Heterogeneity in transformation efficiency of NIH 3T3 cells as determined by transfection with oncogenes

Clones of cells have been isolated from a culture of NIH 3T3 cells and characterized. A high degree of variation between the clones was observed in their efficiency of transformation following transfection with the ras and myc oncogenes. No correlation was found between this characteristic and either the growth rate of these cells in vitro or the efficiency of transfection, as judged by the acquisition of geneticin resistance and chloramphenicol acetyl transferase activity. Continuous maintenance of NIH 3T3 cells in culture resulted in a significant decrease in susceptibility to transformation. It is suggested that the NIH 3T3 cell line comprises a heterogeneous population of cells, and it is the balance between the various types of cells which determines the phenotype of the culture. This balance can be spontaneously disrupted while the culture is grown continuously in vitro.     

NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype.

In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.     

在NIH3T3细胞中高表达EDAG—1基因导致细胞恶性转化

探讨一种新近克隆的特异表达在造血系统组织和细胞中的胚胎发育相关新基因 (EDAG 1)的生物学功能。构建EDAG 1真核表达载体pcDNA3.1( ) EDAG 1,以脂质体法将其稳定转染入NIH3T3细胞中 ,结果发现高表达EDAG 1的NIH3T3细胞失去了正常的平行走向及接触抑制现象 ,侵袭力较正常增强约 10倍并获得锚定不依赖生长能力。将该细胞株皮下接种裸鼠 ,受鼠在 4周左右 10 0 % (6 / 6 )成瘤。这些结果表明EDAG 1是一种具有转化活性的新基因 ,可能与肿瘤发生、发展密切相关     

An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family

We recently reported the cloning of a rearranged human oncogene following transfection of DNA from Kaposi's sarcoma into NIH 3T3 cells. To identify the protein(s) encoded in two novel mRNAs of 3.5 and 1.2 kb expressed in NIH 3T3 transformants, we constructed a cDNA library. One of the cDNA clones isolated (KS3) corresponded to the 1.2 kb mRNA and transformed NIH 3T3 cell when inserted into a mammalian expression vector. The 1152 nucleotide KS3 cDNA encodes a protein of 206 amino acids with significant homology to the growth factors basic FGF and acidic FGF. Expression of the KS3 product as a bacterial fusion protein or in COS cells allowed us to determine that both proteins had significant growth-promoting activity and that the COS cell protein was glycosylated. Thus one of the mRNAs transcribed from the KS oncogene encodes a growth factor that could transform cells by an autocrine mechanism and appears to represent a new member of the FGF family.     

人MCP cDNA在NIH3T3细胞中表达及功能研究

通过构建表达人膜辅因子蛋白ＭＣＰ编码区全长ｃＤＮＡ的重级载体ｐｃＤＮＡ３ＭＣＰ,以磷酸钙沉淀法转染ＮＩＨ３Ｔ３细胞,用Ｇ４１８筛选阳性克隆,并鉴定ＭＣＰ ｃＤＮＡ在细胞中的表达。将血清梯度稀释并与细胞孵育,然后检测细胞活力：灭活的人血清不能引起对照细胞与ＭＣＰ转染细胞的胞毒作用,而新鲜人血清可使对照细胞发生补体依赖的细胞毒反应,ＭＣＰ转染细胞由于ＭＣＰ蛋白的合成,细胞受到保护不发生该反应。另外,Ｍ     

A recombinant murine retrovirus for simian virus 40 large T cDNA transforms mouse fibroblasts to anchorage-independent growth.

A recombinant murine retrovirus containing the intact cDNA sequence for the simian virus 40 (SV40) large T antigen (T) was constructed by using the pZIPNeo SV(X)1 vector. Psi 2 packaging cells were then transfected, and G418-resistant clones were used to generate helper-free viral stocks. NIH 3T3 mouse fibroblasts infected by the recombinant T cDNA retrovirus were selected fro G418 resistance. Such cultures synthesized authentic SV40 T and were transformed to anchorage-independent growth at high efficiency. Therefore, this vector has allowed the study of the transformation properties of T under conditions of neutral drug selection and in the absence of SV40 small t antigen.     

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.     

Cloned hst gene from normal human leukocyte DNA transforms NIH3T3 cells

The hst gene was originally identified as a transforming gene in DNAs from stomach cancers and a noncancerous portion of stomach mucosa by transfection assays using NIH3T3 cells (1,2). Subsequently, the hst gene obtained directly from leukocyte DNA of a leukemia patient was sequenced (3,4). Here, cosmid clones containing the hst gene were isolated directly from normal human leukocyte DNA and from T361-2nd-1 cells, a secondary transformant of NIH3T3 cells induced by transfection of DNA from a stomach cancer. All clones containing the hst gene from these different sources transformed NIH3T3 cells with similar efficiency. Restriction map of the hst gene from normal leukocyte DNA was identical with that from leukocyte DNA of a leukemia patient, while the hst gene from T361-2nd-1 cells was rearranged at the 168th nucleotide upstream of the TATA box.     

Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells

Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14 - 18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CTheta, Src, Bax and mu-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, mu-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells.     

Identification of a transforming gene of human papillomavirus type 16.

Previously, we observed sequential two-step alteration, growth stimulation, and progression to a more malignant state in NIH 3T3 cells transfected by human papillomavirus type 16 (HPV-16) DNA. In this study, we prepared a cDNA library from RNA extracted from cells transfected with the HPV-16 DNA and isolated cDNA clones which had growth-stimulating activity. Analysis of these cDNA clones indicated that the E7 open reading frame alone is responsible for inducing both steps of this cell transformation.     

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.     

Involvement of calpain in melanogenesis of mouse B16 melanoma cells

In the current study, the involvement of calpain, a cysteine proteinase in the regulation of melanogenesis was examined using mouse B16 melanoma cells. In response to α-melanocyte-stimulating hormone (α-MSH), B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. The total calapain activity was decreased within 2 h following α-MSH-treatment, and restored to the initial level in 6–12 h. To further investigate the involvement of calpain in the regulation of melanogenesis, the effect of calpain inhibitors on α-MSH-induced melanogenesis was examined. Inhibition of calpain by either N-acetyl-Leu-Leu-norleucinal (ALLN) or calpastatin (CS) peptide blocked α-MSH-induced melanogenesis. The magnitude of inhibition of melanin biosynthesis was well correlated with a decrease in the activity of tyrosinase, a key regulatory enzyme in melanogenesis. Treatment of B16 cells with ALLN caused marked decrease in both tyrosinase protein and mRNA levels. These results indicate that calpain would be involved in the melanogenic signaling by modulating the expression of tyrosinase in mouse B16melanoma cells.     

Calpain-dependent calpastatin cleavage regulates caspase-3 activation during apoptosis of Jurkat T cells induced by Entamoeba histolytica

In this study, we investigated whether there is a signalling interaction between calpain and caspase-3 during apoptosis in Jurkat T cells by Entamoeba histolytica. When Jurkat cells were co-incubated with E. histolytica, phosphatidylserine externalisation and DNA fragmentation markedly increased compared with results for cells incubated with medium alone. In addition, E. histolytica strongly induced cleavage of caspases-3, -6, -7 and poly(ADP-ribose) polymerase. A rise in intracellular calcium levels and activation of calpain were seen in Jurkat cells after exposure to E. histolytica. Pretreatment of Jurkat cells with calpain inhibitor calpeptin effectively blocked E. histolytica-triggered cleavage of caspase-3 as well as calpain. In contrast, pan-caspase inhibitor did not affect E. histolytica-induced calpain activation. In addition, incubation with E. histolytica resulted in multiple fragmented bands of calpastatin, which is an endogenous inhibitor of calpain, in Jurkat T cells. Moreover, Entamoeba-induced calpastatin degradation was dramatically suppressed by pretreatment with calpeptin, but not by z-VAD-fmk. Entamoeba-induced DNA fragmentation was strongly retarded by z-VAD-fmk, but not calpeptin. Our results suggest that calpain-mediated calpastatin degradation plays a crucial role in regulation of caspase-3 activation during apoptosis of Jurkat T cells by E. histolytica.     

Regulation of tumor invasion and metastasis in protein kinase C epsilon-transformed NIH3T3 fibroblasts

Protein kinase C epsilon is an oncogenic, actin nucleating protein that coordinately regulates changes in cell growth and shape. Cells constitutively expressing PKCepsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions. Here we report that the regulatory C1 domain of PKCepsilon contains an actin binding site that is essential for the formation of elongate invadopodial-like structures, increased pericellular metalloproteinase activity, in vitro invasion of a Matrigel barrier, and the invasion and metastasis of tumors grown in vivo by PKCepsilon-transformed NIH3T3 fibroblasts in nude mice. While removing this small actin binding motif caused a dramatic reversion of tumor invasion, the deletion mutant of PKCepsilon remained oncogenic and tumorigenic in this experimental system. We propose that PKCepsilon directly interacts with actin to stimulate polymerization and the extension of membrane protrusions that transformed NIH3T3 cells use in vivo to penetrate and degrade surrounding tissue boundaries.     

Transformation of NIH 3T3 cells by enhanced PAR expression

Prostate androgen regulated (PAR) is a 1038bp novel gene located on chromosome 1 in epidermal differentiation complex. The gene is ubiquitously expressed in normal tissues and is overexpressed in most of their malignant counterparts. PAR cellular function is unknown. Here we report the effect of increased PAR expression induced by transfection of PAR cDNA on NIH3T3 cell phenotype. PAR-NIH3T3 transfectants expressing 3- to 4-fold higher PAR levels compared to controls grew faster in tissue cultures, formed colonies in soft agar, and exhibited a shortening of G1 and S phases of cell cycle and formed tumors in SCID mice. Transfection of NIH3T3 cells with increased ectopic PAR expression with a 22 mer oligonucleotide in antisense orientation with PAR mRNA abrogated their ability to form colonies in soft agar. The data presented here along with our previously reported results on DU145 cells transfected with antisense PAR cDNA suggest that PAR gene behaves like a proto-oncogene.     

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.     

Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies

Exposure of neu-oncogene-transformed NIH 3T3 cells to monoclonal antibodies reactive with the neu gene product, p185, results in the rapid and reversible loss of both cell-surface and total cellular p185. Although not directly cytotoxic, monoclonal anti-p185 antibody treatment causes neu-transformed NIH 3T3 cells to revert to a nontransformed phenotype, as determined by anchorage-independent growth. Isotype matched control antibodies of an unrelated specificity do not affect p185 levels or colony formation in soft agar by neu-transformed NIH 3T3 cells. Soft agar colony formation by NIH 3T3 cells transformed by ras oncogenes is not affected by anti-p185 antibody treatment. Anchorage-independent growth of cells from the ethylnitrosourea-induced rat neuroblastoma line in which neu was originally detected by DNA transfection is also inhibited in the presence of anti-p185 monoclonal antibodies. Collectively, these results suggest that p185 is required to maintain transformation induced by the neu oncogene.