Human oncogene-transfected tumor cells display differential susceptibility to lysis by lymphokine-activated killer cells (LAK) and natural killer cells

NIH 3T3 tertiary transfectants containing the N-ras or c-Ha-ras oncogenes derived from human tumors were tested for susceptibility to lymphokine-activated killer (LAK) cell and natural killer (NK) cell lysis. N-ras tertiary transfectants contained a human acute lymphocytic leukemia-derived N-ras oncogene. C-Ha-ras transfectants contained either the position 61-activated form of the oncogene (45.342, 45.322, and 45.3B2) or the position 12-activated form (144-162). In 4 hr 51Cr release assays, seven of seven in vivo grown human oncogene transfected NIH 3T3 fibroblasts were lysed by murine LAK effectors, whereas six of seven were lysed by human LAK effectors. There was no difference in susceptibility to lysis between cells transfected with the N-ras oncogene, the position 61 activated c-Ha-ras oncogene, or the position 12 activated c-Ha-ras oncogene. Cultured NIH 3T3 fibroblasts, as well as in vitro and in vivo grown NIH 3T3 tertiary transfectants were resistant to lysis by murine NK effectors and were relatively resistant (4/6 were not lysed) to lysis by human NK effectors. We conclude that human oncogene-transfected tumors are susceptible to lysis by both murine and human LAK cells while being relatively resistant to lysis by murine and human NK cells. Different oncogenes or the same oncogene activated by different point mutations do not specifically determine susceptibility to lysis by LAK or NK. Also the presence of an activated oncogene does not appear to be sufficient for inducing susceptibility to these cytotoxic lymphocyte populations.     

Activated N-ras controls the transformed phenotype of HT1080 human fibrosarcoma cells

To investigate whether the activated N-ras oncogene of HT1080 human fibrosarcoma cells contributes to the expression of the transformed phenotype, we have isolated flat revertants. In two independent revertant lines, an increase in chromosomal ploidy occurred without a concomitant increase in the number of copies of the N-ras transforming allele. Immunoprecipitation confirms that the level of the mutant N-ras p21 gene product in the revertants is correspondingly lower than in HT1080. Analysis of sporadic tumors derived from the revertant cells reveals an increased dosage of the transforming allele. The revertants also retransform after transfection of cloned activated ras oncogenes. These results imply direct participation of an N-ras oncogene in maintaining the transformed phenotype of a human tumor cell line.     

Use of gene transfer and a novel cosmid rescue strategy to isolate transforming sequences.

Mouse Lewis Lung tumor DNA was ligated to a cosmid containing a geneticin (G418)/kanamycin resistance gene and transferred into NIH3T3 cells. Recipient cells were first selected for geneticin resistance and subsequently for their ability to grow as a tumour when injected into nude mice. By repeating this transfection procedure with DNA from resultant tumours, geneticin-resistant NIH3T3 cells were obtained which were tumorigenic and contained approximately 1-5 copies of the transferred cosmid. The functional oncogene was cloned by preparing cosmid libraries of third round tumour DNAs, using a cosmid which does not contain a kanamycin resistance gene. Due to the original linkage of the oncogene with the cosmid containing the kanamycin resistance gene, a series of kanamycin-resistant cosmids were isolated, five of which contained an active oncogene. Subsequent analysis showed that the oncogene present was highly related to the human N-ras gene. Using a DNA probe from the MLL N-ras gene, a non-transforming counterpart was isolated from mouse liver DNA. A comparison between the two N-ras genes showed that a mutation at the amino acid position corresponding to 61 in the human gene is responsible for transforming activity of the rescued gene.     

Carcinogens with diverse mutagenic activities initiate neoplastic guinea pig cells that acquire the same N-ras point mutation

N-ras has been identified by molecular cloning and DNA sequence analysis as the activated oncogene in carcinogen-induced guinea pig transformation. The deduced guinea pig amino acid sequence differs from that of human and mouse by 1 and 4 residues, respectively; the mismatches were in the C-terminal half of the fourth exon. The activated N-ras clone has an AT to TA transversion at the third position of codon 61 which results in the insertion of histidine instead of glutamine. The same activated N-ras gene with the identical mutation was found in all lines regardless of initiating carcinogen (aromatic aryl hydrocarbons or alkylating agents). These results suggest that the mutational event was independent of the mutagenic activity of the initiating carcinogen.     

The HL-60 transforming sequence: a ras oncogene coexisting with altered myc genes in hematopoietic tumors

The oncogene of the HL-60 human promyelocytic leukemia cell line has been passed serially through NIH/3T3 mouse fibroblasts. Oncogene-specific probes prepared from the resulting tertiary transfectants by molecular cloning have been used to show that loss of the transfected oncogene from NIH/3T3 cells correlates with reversion to nontransformed morphology. Analysis of cells transfected by the oncogenes of other tumors and tumor cell lines indicates that the transforming gene of the HL-60 leukemia cell line is closely related to oncogenes of a Burkitt's lymphoma, an acute myelogenous leukemia, an adenocarcinoma of the colon, a neuroblastoma, and two sarcomas. This oncogene is distantly related to the viral oncogenes of Kirsten and Harvey sarcoma viruses. It has been termed N-ras. The active N-ras oncogene coexists with altered versions of the myc oncogene in the HL-60 and AW Ramos human tumors. This suggests a multistep mechanism involving both ras and myc genes in the creation of these tumors.     

Oncogenicity of human N-ras oncogene and proto-oncogene introduced into retroviral vectors.

The N-ras gene is the only member of the ras family which has never been naturally transduced into a retrovirus. In order to study the in vitro and in vivo oncogenicity of N-ras and to compare its pathogenicity to that of H-ras, we have inserted an activated or a normal form of human N-ras cDNA into a slightly modified Harvey murine sarcoma virus-derived vector in which the H-ras p21 coding region had been deleted. The resulting constructions were transfected into NIH 3T3 cells. The activated N-ras-containing construct (HSN) induced 10(4) foci per microgram of DNA and was found to be as transforming as H-ras was. After infection of the transfected cells by either the ecotropic Moloney murine leukemia virus or the amphotropic 4070A helper viruses, rescued transforming viruses were injected into newborn mice. Both pseudotypes of HSN virus containing activated N-ras induced the typical Harvey disease with similar latency. However, we found that the virus which contained normal N-ras p21 (HSn) was also pathogenic and induced splenomegaly, lymphadenopathies, and sarcoma in mice after a latency of 3 to 7 weeks. In addition, Moloney murine leukemia virus pseudotypes of N-ras caused neurological disorders in 30% of the infected animals. These results differed markedly from those of previous experiments in which we had inserted the activated form of N-ras in the pSV(X) vector: the resulting SVN-ras virus was transforming on NIH 3T3 cells but was poorly oncogenic in vivo (M. Souyri, C. F. Koehne, P. V. O'Donnel, T. H. Aldrich, M. E. Furth, and E. Fleissner, Virology 158:69-78). However, similarly poor oncogenicity was also observed when the v-H-ras coding sequence was inserted in pSV(X) vector, which indicated that the vector sequences play a crucial role in the pathogenicity of a given oncogene. Altogether, these data demonstrated unequivocally that N-ras is potentially as oncogenic as H-ras and that such oncogenic effect could depend on the vector environment.     

Ras (proto)oncogene induces N-linked carbohydrate modification: temporal relationship with induction of invasive potential

The effect of expression of the ras oncogene on protein glycosylation was studied. VSV G-protein and class I histocompatibility antigens were analysed to monitor ras-mediated changes in glycosylation. Transient expression of the c-Ha-ras oncogene, introduced into NIH 3T3 cells by the DEAE-dextran method, altered protein glycosylation within 25 h of transfection. The same result was obtained after dexamethasone-induced expression of p21-ras in stable NIH 3T3 transfectants containing either an activated Ha-ras oncogene or a normal N-ras proto-oncogene under control of the glucocorticoid-inducible MMTV promoter. The alteration of cell surface carbohydrates, induced by the ras (proto)oncogene and the subsequent acquisition of invasive potential, occurred prior to morphological transformation.     

The N-ras oncogene assigned to the short arm of human chromosome 1

The human N-ras oncogene, isolated from the HL-60 promyelocytic leukemia cell line, is distantly related to viral oncogenes of Kirsten and Harvey sarcoma viruses. We have determined its chromosomal location by Southern blot analysis of DNAs from 37 human x rodent hybrid cell lines derived from 8 different human donors, some of whom carried balanced rearrangements of chromosome 1. The results indicate that the N-ras oncogene (RASN) is localized on the proximal part of the short arm of human chromosome 1, in region p3200 leads to cen.     

Highly frequent detection of transforming genes in acute leukemias by transfection using in vivo selection assays

DNAs from nine out of ten acute leukemia cases that were negative by in vitro focus forming assays exhibited transforming activity tested by in vivo selection assays in nude mice using transfected NIH3T3 cells. Of the nine cases, six cases contained activated N-ras genes, and one case exhibited activation of the c-K-ras gene. None of the ras gene family showed homology with the transforming genes derived from the other two cases. Our observations indicate that in vivo selection assays detect transforming genes including ras oncogenes at high frequency, and that activated N-ras genes are frequently detected in human acute leukemias.     

Activation of N-ras in a human melanoma cell line.

DNA isolated from cell line Mel Swift, a human melanoma cell line, transforms NIH3T3 cells. Southern blot analysis of DNA from secondary foci revealed conserved 8.8- and 7.8-kilobase EcoRI fragments which hybridized with a human repetitive sequence clone, blur 8. The activated transforming gene was identified as N-ras, and the 8.8-kilobase EcoRI fragment from a secondary transformant was cloned. Synthetic 17-mer oligonucleotides which spanned either the normal codon 61 (CAA) or a mutant codon 61 (AAA) were used for hybridization. Cloned N-ras from melanoma cell line Mel Swift hybridized to the mutant (AAA) oligonucleotide. From this we predicted a glutamine-to-lysine substitution in amino acid 61, a change confirmed by conventional sequencing of the first and second exons of N-ras from cell line Mel Swift. Transfection experiments showed that only those recombinant clones with the mutation in position 61 were biologically active.     

Inhibition of MAPK activity, cell proliferation, and anchorage-independent growth by N-Ras antisense in an N-ras-transformed human cell line

Mammalian ras genes encode a family of plasma membrane-bound proteins that function as intermediates in signal transduction pathways involved in cell growth and differentiation. Ras oncogene is frequently involved in neoplastic transformation of different cellular histotypes. In this study, we tested the ability of antisense oligodeoxyribonucleotides (AS-ODN) that have mixed phosphodiester/phosphorothioate backbone, targeted against human N-Ras, to inhibit N-ras gene expression and to specifically interfere with the Ras-dependent activity of mitogen-activated protein kinase (MAPK) in two human cell lines carrying an endogenous N-ras mutated allele at codon 61. Three AS-ODN that inhibit basal MAPK activity have been identified. Moreover, AS-ODN treatment resulted in potent antiproliferative effects in cell culture and great inhibition of N-ras mRNA levels in one of two cell lines. These studies suggest that antisense molecules, targeted against N-Ras, could be of considerable value as a tool to study the N-Ras-specific transduction pathway.     

Modulatory effect of environmental endocrine disruptors on N-ras oncogene expression in the hermaphroditic fish, Kryptolebias marmoratus

Kryptolebias marmoratus is the only known internally self-fertilizing vertebrate. It shows high susceptibility to many chemical carcinogens and has been proposed as a potential cancer model species alternative to mammals. Since use of this fish species is expected to rise in cancer research, regulation of oncogenes from K. marmoratus needs proper understanding. We cloned and deduced full-length sequence of cDNA of N-ras oncogene from K. marmoratus. Study of expression profile of N-ras by using quantitative real-time RT-PCR revealed that brain had the highest level of expression compared to other tissues. Some embryonic stages showed more N-ras expression than juveniles and adults. Exposure to two environmental endocrine disrupting chemicals (EDCs), bisphenol A (BPA) and 4-nonylphenyl (NP) caused up-regulation of N-ras in gonad, intestine and liver of hermaphrodite K. marmoratus. It is suggested that K. marmoratus may be a suitable model species for oncogene expression studies. The observed EDC-induced expression of N-ras supports the assumption that EDC exposure may predispose the host to the risk of environmental carcinogenesis.     

维生素E和微量元素Se对人肝癌细胞生长、分化及其癌基因表达的影响

本工作观察了体外环境中不同水平的维生素Ｅ和微量元素Ｓｅ对人肝癌细胞株（ＳＭＭＣ－７７２１）生长、分化和其癌基因（Ｎ－ｒａｓ、ｃ－ｍｙｃ）表达水平的影响。实验结果表明：高水平维生素Ｅ（２．４、９．２、２４．０ｎｍｏｌ／Ｌ）和Ｓｅ（０．１５、０．３０、０．６０ｎｍｏｌ／Ｌ）对肝癌细胞的集落形成率具有明显的抑制作用;生化分析显示高水平维生素Ｅ和微量元素Ｓｅ均可明显抑制环境中脂质过氧化的水平,Ｓｅ对癌细胞甲胎蛋白的分泌有明显的抑制作用,而维生素Ｅ作用不明显。细胞原位杂交发现维生素Ｅ浓度为２．４和９．２ｎｍｏ１／Ｌ时对细胞癌基因Ｎ－ｒａｓ的表达具有明显抑制作用;Ｓｅ浓度为０．１５和０．３０ｎｍｏｌ／Ｌ时对癌基因ｃ－ｍｙｃ的表达明显抑制。实验还观察了维生素Ｅ和Ｓｅ之间的叠加效应,结果显示除对环境中脂质过氧化的抑制作用具有叠加效果外,对其他指标没有明显作用。     

外源性神经节苷脂GM_3影响人肝癌细胞生长的可能机制的初步研究

通过苔酚蓝染色细胞发现,外源性GM3（10μg/ml）能明显抑制人肝癌细胞株SMMC-7721细胞生长,在GM3处理3d时,出现明显差异．通过NorthernBlot分析发现,外源性GM3可明显影响人肝癌细胞株SMMC-7721细胞中c-fos、c-jun、c-myc和N-ras这四种癌基因的mRAN表达．未经GM3处理的细胞中没有检测到c-fosmRNA,但c-jun微量表达,并有c-myc和N-rasmRNA的高水平表达而GM3可在短时间内快速大量地诱导c-fos、c-junmRNA的生成．GM3处理的细胞,c-myc和N-rasmRNA的表达均明显减少．GM3处理45min时,c-myc基因表达只为对照组的39．55％;GM3处理24h时,N-ras基因表达为对照组的30.48％．以上结果提示：GM3抑制SMMC-7721细胞生长很可能是通过改变癌基因表达来实现的．     

人N-ras癌基因细胞型特异DNA结合蛋白

本文利用Southwestern印迹技术发现人肿瘤HT1080细胞染色质蛋白中一组与N-ras基因结合的蛋白,分子量约为150,105,95,90KDa,而与Ha-ras基因结合的一组蛋白,分子量约为160,115,100,55KDa,其中150KDa蛋白是N-ras基因特异的DNA结合蛋白,具有细胞型特异性,在HT1080细胞中含量最多,T24细胞次之,而在人HeLa细胞,淋巴细胞、肠细胞以及未转化的NTH3T3细胞中未被发现。此种蛋白可能与N-ras基因在HT1080细胞内的激活有密切关系。     

Mechanism of activation of an N-ras gene in the human fibrosarcoma cell line HT1080.

A full length N-ras gene has been cloned from both the human fibrosarcoma cell line HT1080 and from normal human DNA. N-ras isolated from HT1080 will efficiently induce morphological transformation of NIH/3T3 cells in a transfection assay, whereas N-ras isolated from normal human DNA has no effect on NIH/3T3 cells. The coding regions of the normal N-ras gene have been sequenced and the predicted amino acid sequence of the N-ras product is very similar to that of the c-Ha-ras1 and c-Ki-ras2 products. By making chimeric molecules between the two cloned genes the activating alteration in the HT1080 N-ras gene has been localised to a single base change that results in an amino acid alteration at position 61 of the p21 N-ras product.     

人类乳腺癌中几种癌基因的状态与临床指征关系的初步探讨

本文报告了乳腺癌标本中N-ras、c-myc和neu癌基因异常改变,包括扩增和重排,并对这些结构改变与乳腺癌临床指征的关系作了初步的分析。本文的实验结果表明这些癌基因可能在人类乳腺癌的生物学行为和发病方面起着一定的作用。