Correlation of growth capacity of cells in hard agarose with successful transfection by the activated c-Ha-ras oncogene and in vivo proliferative capacity at metastatic sites

The purpose of this study was to determine whether the degree of anchorage-independent growth of rodent or human cells in increasing concentrations of agarose correlated with successful transfection of the cells with an activated c-Ha-ras oncogene and tumorigenicity in nude mice. NIH 3T3 cells, C3H 10T1/2 fibroblasts, four clones of the murine K-1735 melanoma with different metastatic capacities and the TE85 human osteogenic sarcoma line were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, which confers resistance to neomycin (pSV2-neoEJ). Cells transfected with pSV2-neo, a plasmid containing the neo gene, served as controls. Cells from parental or transfected lines (selected by Geneticin) were plated into medium containing 0.3%, 0.6% 0.9%, or 1.2% agarose. These cells were also injected subcutaneously and intravenously into nude mice. The production of tumor cell colonies in dense agarose (greater than or equal to 0.6%) correlated with successful transfection with pSV2-neoEJ and production of experimental metastases in the lung of nude mice. We conclude that the degree of anchorage-independent growth of cells predicts successful transfection with activated c-Ha-ras oncogene and tumorigenic behavior in vivo. Thus this technique may be useful for the detection of cells transfected with transforming oncogenes.     

Activated N-ras oncogene in a transformant derived from a rat small intestinal adenocarcinoma induced by 2-aminodipyrido[1,2-a:3',2'-d]imidazole

DNAs from five intestinal adenocarcinomas induced by 2-aminodipyrido[1,2-a:3',2'-d]imidazole,which is present in broiled fish, were subjected to transfectionassay using NIH3T3 cells as recipients. The DNA from only oneadenocarcinoma induced a morphologically transformed focus.Rat N-ras sequences were detected in the primary transformantand in three tested secondary transformants. In the activatedN-ras oncogene, a G—T transversion at the first letterof codon 12 was detected. The original tumor DNA did not hybridizewith the oligonucleotide representing the mutated allele, butdid hybridize with the one representing the normal allele. Fromthese data we concluded either that the activation of the N-rasoncogene had occurred during the transfection or that the activatedN-ras oncogene had been present in a minor population of cellsin the original tumor.     

Activation of H-ras oncogene in 3-methylcholanthrene-transformed human cell line

DNA prepared from the 3-methylcholanthrene (3MC)-transformedhuman 312H cell line induced foci on NIH/3T3 cells, whereasDNAs prepared from 7,12-dimethylbenz[a]-anthracene-transformedand the dimethylsulfoxide control 312H cell lines failed toinduce foci. The transformed gene from the 3MC-transformed 312Hcells was identified as an activated form of the human cellulartransforming H-ras oncogene. Analysis of the ras oncogene p21product in this transformant by immunoprecipitation and gelelectrophoresis suggested that this gene was activated by mutationin the 61st codon. These findings demonstrate that activationof a member of the ras gene family can occur in a chemicallytransformed human cell line.     

Metastatic potential of cloned murine melanoma cells transfected with activated c-Ha-ras

We sought to determine whether the transfection of tumorigenic but not metastatic cells with the activated c-Ha-ras oncogene was invariably associated with acquisition of the metastatic phenotype. Three clonally derived lines of the K-1735 murine melanoma, characterized as nonmetastatic or poorly metastatic, were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, that confers resistance to neomycin (pSV2neoEJ). Cells transfected with pSV2neo, a plasmid containing the neo gene, served as controls for the procedure of Polybrene-mediated transfection. All cell lines were injected into syngeneic C3H/HeN and into athymic mice, and the results were compared with those produced by highly metastatic K-1735 M-2 cells. Although the pSV2neoEJ-transfected cells produced more rapidly growing s.c. tumors than the control cell lines did, the incidence of spontaneous metastasis was not increased. Following i.v. inoculation, the c-Ha-ras transfectants were retained in lung vasculature in greater proportions than pSV2neo counterpart transfectants were. The c-Ha-ras transfectants also produced significantly more lung tumor colonies, which grew faster than the few lung tumor colonies in mice given injections of control melanoma cells. We concluded that transfection of the activated c-Ha-ras oncogene into nonmetastatic K-1735 melanoma cells leads to accelerated tumor growth in vivo and can confer the ability to form lung colonies after i.v. injection but not the ability to metastasize from a primary s.c. tumor.     

Detection of distinct transforming genes in X-ray induced tumors

DNAs from mouse skin tumors (papillomas, squamous cell carcinomas,basal cell carcinomas and pilomatrixomas) initiated with X-irradiationand promoted with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)demonstrated dominant transforming activity by the productionof transformed foci in the mouse recipient tine, NIH3T3. Dominanttransforming activity was not found in DNA isolated from normalmouse epidermis or from the corresponding liver. The NIH3T3transformants induced with squamous cell carcinoma DNA grewin soft agar and formed tumors in nude mice. Southern blot analysisof primary NIH3T3 transformant DNAs carrying oncogenes fromradiation-initiated squamous cell carcinomas indicated thatthe oncogenes responsible for the transformation of the recipientcells were not Ha-ras, Ki-ras or N-ras genes, nor were theyerbB, B-lym, met, neu or raf. The data presented indicate thatDNAs from radiation-initiated mouse skin tumors contain dominanttransforming genes that are detectable by DNA-mediated genetransfer. The oncogene sequences activated in these radiation-initiatedtumors are distinct non-ras transforming genes.     

Direct tumorigenic conversion of human gallbladder carcinoma cells by v-src but not by activated c-H-ras oncogene

The roles of activated ras and src oncogene products in the acquisition of fully neoplastic phenotype by human gallbladder adenocarcinoma cells were investigated by co-transfecting non-tumorigenic HAG-1 human gallbladder carcinoma cells with the pSV2neo plasmid and a plasmid carrying either activated c-H-ras or v-src oncogene. G-418-resistant clones were isolated and assessed for the acquisition of anchorage-independent growth potential. Neither the 10 established clones transfected with pSV2neo alone nor the 17 clones transfected with activated c-H-ras, including 4 clones expressing the mutated p21^H-ras protein, could form colonies in soft agar. By contrast, out of 10 clones transfected with v-src, 2 formed colonies in soft agar and produced tumors in athymic nude mice, the resulting progressive neoplasms being poorly differentiated adenocarcinomas. These tumorigenic clones were shown to have v-src DNA and mRNA levels with p60^v-src protein, but there were no significant chromosomal alterations following tumorigenic conversion. Moreover, herbimycin A, a selective src-kinase inhibitor, markedly reduced clonogenic growth of these cells in soft agar rather than monolayer growth, suggesting that anchorage-independent growth of the v-src-transformed HAG-1 cells might be driven directly by p60^v-src kinase activity. Taken together, our data suggest that the fully neoplastic conversion of HAG-1 cells depends on src-related tyrosine-kinase activity, but not solely on the function mediated by activated ras, thus providing evidence of an src-related signaling pathway for the acquisition of tumorigenic potential by human gallbladder adenocarcinoma cells. © 1995 Wiley-Liss, Inc.     

Morphological transformation of immortalized hamster dermal fibroblasts following treatment with simple alkylating carcinogens

We have examined the mechanism of transformation of a line ofimmortalized hamster dermal fibroblasts (4DH2 cells) followingtreatment with the simple alkylating agents, N-methyl-N-nitrosourea(MNU), N-ethyl-N-nitrosourea (ENU) and dimethyl sulphate (DMS).Treatment of 4DH2 cells with the potent point mutagens MNU andENU gave rise to a spectrum of foci of different sizes, includingprogressively growing large foci and compact small foci. Incontrast, treatment with the weak point mutagen DMS producedmostly large foci. The ability of cell lines derived from morphologicallytransformed foci to grow in soft agar in general reflects theiroriginal size. Thus most cell lines derived from large focigrew in soft agar while most lines derived from small foci didnot. Transfection of cellular DNAs into the parent 4DH2 cellline and into NIH3T3 mouse fibroblasts has revealed the presenceof dominantly acting transforming genes in the chemically transformedcell lines. Thus DNA from five of six cell lines derived byculturing large foci and from one of three cell lines derivedby culturing small foci induced efficient morphological transformationof the recipient cells. Southern analyses of DNA from primaryand secondary transfectants showed that several of the transforminggenes transferred in these experiments were not closely relatedto H-ras, K-ras or N-ras     

The rat N-ras gene; interference of pseudogenes with the detection of activating point mutations

The PCR technique in combination with selective hybridizationto mutation specific oligonucleotides, is a widely used methodologyfor the detection of activating point mutations in ras oncogenes.In the present paper we demonstrate for the N-ras gene of therat that processed pseudogenes do interfere with this method.A first indication for this interference came from the sequenceanalysis of cloned PCR fragments of exon 1, amplified with primersderived from previously reported exon sequences of the mouseN-ras gene. Between different clones originating from one PCRreaction, a marked sequence heterogeneity is observed and thisis shown to be the result of the presence of at least two differentprocessed pseudogenes of the rat N-ras gene. These two pseudogenes,together with the wildtype N-ras gene and a small 3' part ofthe unr gene, were eventually cloned and their genomic organizationand nucleotide sequences determined. Furthermore, representativeexamples of the confounding effects of these pseudogenes onthe screening for activating point mutations are presented.Taken together, our results demonstrate that intron-speceficamplification is a prerequisite for the unambiguous detectionof activating point mutations in the N-ras gene of the rat.     

Different malignant phenotypes induced in a stable, subdiploid, benign epithelial clone by DNA transfection.

Progression to malignancy in carcinomas has been studied in a stable, benign, subdiploid, cloned epithelial cell line (A5P/B10) sensitive to Geneticin at 100 micrograms/ml. A total of 28 cell lines were selected for Geneticin - resistance and inoculated into the footpads of syngeneic animals following co-transfection with pSV2neo and genomic DNA, or transfection with plasmid constructs containing neo and the activated Ha-ras oncogene. The behavior of 12 cell lines cotransfected with normal genomic DNA and inoculated into 146 footpads was the same as the A5P/B10 cells. Low grade primary tumors were produced in 122 footpads by 13 cell lines transfected with Ha-ras, and a proportion (61/122) produced well-differentiated lymph node metastases. One of 3 cell lines cotransfected with genomic DNA from a malignant cell line (BC1) produced 8 anaplastic primary tumors with anaplastic metastases. Cell lines from lymph nodes involved by these anaplastic tumors were sensitive to Geneticin, and genomic DNA from 2 clones of these cells failed to produce a malignant phenotype when co-transfected into the A5P/B10 cells. These results indicated that the progression to a malignant phenotype induced in benign cells from a spontaneous epithelial tumor by co-transfection with genomic DNA from malignant cells was different from that induced by the ras oncogene.     

Transformation of human epidermal keratinocytes with fission neutrons

The biological effects of exposures to high LET radiations haveparticular relevance to radiation protection and risk assessmentSince most cancers are of epithelial origin, it is importantto obtain a better understanding of radiationinduced oncogenictransformation in this cell type. Accordingly we have initiatedstudies to determine whether immortalized human epidermal keratinocytes(RHEK) can be transformed with high LET radiations. Exponentiallygrowing RHEK cells were treated with single doses (1, 10, 25,50 and 100 cGy) of 0.85 MeV fission neutrons from the Janusreactor. Neutron exposure led to the development of morphologicallyaltered cells and foci formation after 6 weeks at confluence.These transformed cultures grew with an increased saturationdensity, exhibited anchorageindependent growth and formed tumorshi athymlc mice. Single-strand conformatlonal polymorphism analysisand DNA sequencing demonstrated the absence of point mutationsin codons 12/13 and 61 in the Ha-ras, Ki-ras, or N-ras genesand exons 4–9 of the pS3 tumor suppressor gene. Thesestudies demonstrate that high LET radiations (fission neutrons)can transform immortalized human epithelial cells to a malignantphenotype that does notappear to involve mutations in eitherthe cellular p53 or ras genes.     

Immunohistochemical expression of N-ras oncogene is a late event in head and neck carcinomas

This study investigated the expression of the N-ras oncogene in routinely processed tissue sections from 133 patients with squamous cell carcinoma of the head and neck (SCCHN) by immunohistochemistryusing anti-N-ras monoclonal antibody. N-ras expression was present in 67 of 133 (49.67%) cases. There was a highly significant correlation between N-ras expression and clinical stage of disease (P=0.003). This study confirmed that overexpression of the N-ras oncogene is common in SCCHN and that it may be an important event in the late stage of disease.     

Variant mutational activation of the K-ras oncogene in renal mesenchymal tumors induced in newborn F344 rats by methyl(methoxymethyl)nitrosamine

Renal mesenchymal tumors were induced at high incidence in F344rats by a single intraperitoneal injection of methyl(methoxymethyl)nitrosamine(DMN-OMe) within 48 h after birth. DNAs from 18 of 35 mesenchymaltumors contained transforming ras sequences in NIH3T3 transfectionassays: K-ras (17/18) or N-ras (1/18). Single-stranded conformationalpolymorphism analysis or dideoxy sequencing of polymerase chainreaction-amplified K-ras gene fragments revealed that theseneoplasms contained a variety of activating mutations in theK-ras oncogene. Alterations in codon 12 predominated and includedGGT GAT transitions, GGT GTT or TGT transversions, and previouslyreported insertion mutations, although some tumors expressedmore than one mutation and the pattern of mutations even variedwithin tumors. Mutations were also found in exons 2 and 3. Inaddition, tumor trans-plantability into syngeneic hosts correlatedpositively and significantly with K-ras activation. Renal mesenchymaltumors with transforming mutations in exon 1 were often successfullypassaged (10/12) while tumors which lacked mutations in exon1 were infrequently transplantable (2/14). While the observedbase substitutions in K-ras are consistent with adduct formation,the presence of insertion mutations and intratumor heterogeneityof alterations suggest that ras activation in DMN-OMe-inducedtumors is not necessarily an early event in tumorigenesis.     

N-ras oncogene expression changes the growth characteristics of human melanoma in two independent SCID-hu mouse models

Fifteen percent of all human melanomas carry mutations in ras genes, the majority of which are located in codon 61 of the N-ras gene. However, the biological significance of these mutations is as yet unknown. In this study, we investigated the influence of N-ras oncogene products mutated in codon 61 on the growth characteristics of human melanoma in vivo by establishing 2 SCID-hu mouse xenotransplantation models. Tumors grown in SCID mice injected with human melanoma carrying activated N-ras genes were significantly larger (p < 0.004) than tumors grown in animals injected with the appropriate control transfectants. Additionally, tumors with N-ras point mutations clearly showed a more pleomorphic phenotype than the control groups. Our results, obtained in 2 independent SCID-hu xenotransplantation models, suggest that mutated N-ras oncogene expression may be an important factor influencing growth characteristics of human melanoma without altering metastatic potential. These novel in vivo model systems provide a tool for further study of the biology of mutated ras in melanoma and should also prove useful for testing new and improved treatment strategies for human melanoma carrying mutated ras genes. © 1996 Wiley-Liss, Inc.     

Effects of pseudofype retrovirus containing human N-RAS antisense gene on the growth of human liver cancer LTNM4 transplanted in nude mice

An amphotropic pseudotype retrovirus containing human N-ras antisense gene was constructed and packaged with helper cells. It has been previously demonstrated that the virus did inhibit the growth of human hepatocarcinoma cell line PLC/PRF/5in vitro accompanied with the blockage of p21 expression. Based on these results, further study was carried on to examine the effect of these viruses on the growth of human hepatoma transplanted LTNM4 in nude mice. It has been shown that the retrovirus containing human antisense N-ras gene could inhibit the hepatoma in nude mice at a rate of 78% (P<0.05) as compared with saline control. No inhibition was observed in group treated with retrovirus which contained no N-ras sequence. These resultsin vivo lend further support that human N-ras antisense gene mediated by retrovirus could block the expression of the relevant oncogene and lead to the inhibition of cancer growth. It also provided the basis for further approaches of gene therapy for human cancer.     

Diacylglycerol is an effector of the clonal expansion of cells containing activated Ha-ras genes

Diacylglycerols (DAG) are lipid second messengers which aregenerated during phospholipase-catalyzed hydrolysis of phospholipids.The model DAG, sn-1, 2-didecanoylglycerol (DIC₁₀), is an effectivetopical tumor promoter in 7, 12-dimethylbenz[a]anthracene (DMBA)-initiatedmouse skin. We now report that 11/12 of DMBA-initiated/DIC₁₀-promotedpapillomas examined contain an A — T mutation in the 61stcodon of the Ha-ras gene, suggesting that DAGs affect the clonalexpansion of activated Ha-ras-containing cells. To explore furtherthe DIC₁₀-induced clonal expansion of activated Ha-ras-containingcells, we have examined the tumor-promoting effect of DIC₁₀in the skin of transgenic TG.AC mice, which harbor a v-Ha-rastransgene. By 9 weeks of promotion, 100% of the TG.AC mice developedsquamous papillomas and by 15 weeks these mice developed>20papillomas/mouse. Because fatty acids are known to participatein signal transduction pathways, and since cellular lipasescould cleave the fatty acid side chains present in DIC₁₀, wehave examined the tumor promoting activity of n-decanoic acidto verify the specificity of promotional activity of DIC₁₀.n-Decanoic acid did not function as a tumor promoter. Thesedata implicate DAG as an effector of the clonal expansion ofmutated Ha-ras-containing cells, and support a mechanism wherebyan increase in endogenous DAG could contribute to the clonalexpansion of cells containing a Ha-ras oncogene.     

Transfection of a non-metastatic diploid rat mammary epithelial cell line with the oncogenes for EJ-ras-1 and polyoma large T antigen

Transfection of rat mammary (Rama) 37 epithelial cells which yield non-metastasizing adenomas in syngeneic Wistar-Furth rats with a drug resistance plasmid containing both the neo gene and EJ-ras-1 (pSV2neo.ras) or with pSV2neo and a plasmid encoding the large T Antigen (pLT214) of polyoma virus yields drug-resistant transformants with a frequency of 10(-5). Representative transformants have been propagated in neo-selecting medium to yield various cell lines. The 7 lines transfected with pSV2neo.ras (EJ1 set) and the 10 lines co-transfected with pSV2neo and pLT214 (LT1 set) all produce tumours at subcutaneous (s.c.) sites with a shorter median latent period than tumours produced by the parental Rama 37 cells. In addition, the LT1 set of transformants yields a higher incidence of tumours than the Rama 37 cells. No metastases are produced when any of the oncogene transformants are inoculated s.c. into rats. However, when an EJ1 representative is inoculated intravenously (i.v.), tumour deposits are found in the lungs of the host animals. In contrast, other Rama 37 variants that metastasize from s.c. sites fail to produce any metastases when inoculated i.v. The oncogene transfectants contain integrated DNA that hybridizes to neo and to the requisite oncogenic DNAs; the pattern of hybridizing bands to the transfected genes and their expression as mRNA is complex, and is presented in detail.     

High frequency of c-K-ras activation in 3-methylcholanthrene-induced mouse thymomas

Thymic lymphomas were induced in RJF mice by percutaneous applicationof 3-methykholanthrene (MCA). DNAs from 83% of the tumors analyzedcontained a transforming c-K-ras gene. The high frequency ofc-K-ras oncogene activation in response to MCA seems to favorthe concept that the activation of c-K-ras is related to thespecificity of the mutagenic effect of MCA.     

Analysis of activated protooncogenes in B6C3F1 mouse liver tumors induced by ciprofibrate, a potent peroxisome proliferator

Liver tumors from B6C3F1 mice induced by the potent peroxisomeproliferator ciprofibrate, a hypolipidemic drug, were evaluatedfor the presence of transforming genes by the nude mouse tumorigenicityassay. As reported earlier, the tumors were not activated bya point mutation in codon 61 of H-ras. Two of the eight tumorsexamined contained a mutation in codon 13 or an H-ras gene mutatedin codon 117. Screening of another 23 ciprofibrate-induced livertumors by oligonucleotide hybridization analysis and directDNA sequencing resulted in the identification of three tumorDNA samples with point mutations in codon 117 of the H-ras gene.In addition, another tumor sample contained a K-ras gene witha mutation in codon 61. Mutations in these codons have beenseen only rarely in chemically induced liver tumors from thismouse strain. Of 15 spontaneous B6C3F1 liver tumors screenedin the same manner, one exhibited a K-ras gene activated bya mutation in codon 13 and a second contained an H-ras geneactivated by a mutation in codon 117. These ras gene mutationshave not been reported previously from spontaneous liver tumors.The frequency and spectrum of ras oncogene mutations characterizedin ciprofibrate-induced liver tumors differ significantly fromthe frequency and pattern identified in spontaneously occurringliver tumors. The results of this study with a limited numberof samples suggest that ras protooncogene activation or activationof other protooncogenes that can be detected by the nude mousetumorigenicity assay are not frequent events in the mechanismof carcinogenicity of the peroxisome proliferator ciprofibrate.However, the lower frequency and distinct pattern of H-ras mutationsobserved in these tumors disprove the assumption of promotionof spontaneous hepatocarcinogenesis by ciprofibrate.     

c—myc反义RNA转录体的构建及其对HL60细胞恶性表型的影响

本研究构建了c—myc反义RNA真核细胞可诱导表达质粒PGXC．用PGXC及PSV2neo质粒共转染入早幼粒细胞白HL60，经G418抗性筛选得到转染细胞HL60^R，与亲本细胞相比其生长速率明显减低：细胞周期中G1期细胞增多，而S期细胞相应减少；细胞形态及功能呈现分化诱导；在软琼脂上不能形成集落，该细胞在裸鼠体内的致瘤性亦显著减弱以至消失。