Role of the oncogene in the mutagenic activity of bovine adenovirus type 3

The mutagenic and carcinogenic effect of two EcoRI-fragments of bovine adenovirus type 3 (BAV-3) DNA inserted into pBR325 has been studied. The C fragment (located between 3,6 and 19,7 map units) contains the viral oncogene, the C fragment (between 44,3 and 63,7 map units) displays no transforming activity. It has been established that oncogene BAV-3 statistically true increases the yield of mutants resistant to 6-mercaptopurine (6MP) in Chinese hamster cells. The C fragment, pBR325 without viral sequences and DNA fragments of different molecular weights from normal Syrian hamster cells have no mutagenic effect. The control over tumor formation in syngenic mice after injection of C3H10T 1/2 and D. C fragments and pBR325 treatment exposed a parallelism between the mutagenic and transforming effect. The study of the combined effect of viral DNA fragments and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) which increases the transforming activity of different carcinogens, shows that the promoter increases the frequency of mutants after viral oncogene treatment and does not induce mutagenic activity of those types of DNA which are unable to transform the cells.     

Oncogen localization in the DNA composition of simian adenoviruses. II. The identification in SV20 virus DNA of a specific fragment possessing transforming activity

Simian adenovirus 20 DNA was specifically cleavered by restriction endonucleases EcoRI, BamHI, XbaI and HindIII. The transformation activity of the DNA digest was investigated. BamHI, XbaI, and HindII DNA digests were able to transform the primary rat kidney cell culture (Wistar) as well as the native SV20 DNA. The transforming activity was revealed in a specific fragment of the viral DNA, obtained after the treatment of the DNA with BamHI (fragment B), with molecular weight 5.4 x 10(6) dalton. This fragment is located in the left end of the viral genome. The lack of cell transformation by the EcoRI-hydrolysate of viral DNA may serve a proof of the extremely left position of the oncogene in the viral genome, since of EcoRI-fragment chips off a fragment with molecular weight 3 x 10(5) dalton fr om the left side of DNA molecule.     

Simultaneous transfer of tumorigenic and metastatic phenotypes by transfection with genomic DNA from a human cutaneous squamous cell carcinoma

High-molecular-weight genomic DNA isolated from a human cutaneous squamous cell carcinoma (AS) was assayed for its ability to induce tumorigenic transformation of NIH 3T3 cells. Subcutaneous injection of NIH 3T3 cells cotransfected with DNAs from AS tumor and pSV2-neo plasmid not only induced tumors at the site of injection, but also metastasized spontaneously to the lungs in 100% of nude mice injected. DNA isolated from a representative primary tumor and a metastasis was again used in a second round of transfection. Injection of secondary transfectants into nude mice again resulted in induction of both subcutaneous tumors and spontaneous long metastases. Southern blot hybridization with ras-specific probes revealed that DNA from both primary tumors and metastases induced by AS tumor DNA contained highly amplified Ha-ras oncogene. Furthermore, DNAs from secondary tumors and metastases induced by DNA from a primary tumor and a metastasis also contained similar highly amplified Ha-ras oncogene. These results suggest that the amplified Ha-ras oncogene may be responsible for induction of both tumorigenic and metastatic phenotypes in NIH 3T3 cells transfected with DNA from AS tumor.     

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.     

Transforming genes in human tumors

DNAs isolated from a variety of human tumor cell lines as well as from naturally occurring human carcinomas and sarcomas were shown to induce morphologic transformation upon transfection into NIH/3T3 cells. All tested transformants contained human DNA sequences, some of which specifically cosegregated with the malignant phenotype in additional cycles of transfection. Southern blot analysis of second cycle transformants derived from T24 human bladder carcinoma cells showed the presence of a single 15 kbp EcoRI fragment of human DNA. These sequences were molecularly cloned utilizing λ Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated λ T24-15A, was shown to contain an internal 6.6 kbp Bam HI fragment of human DNA that transformed NIH/3T3 fibroblasts with a specific activity of 5 × 10⁴ focus forming units per picomol. These results indicate that we have moleculary cloned an oncogene present in T24 bladder carcinoma cells. Comparison of molecular clones containing the T24 oncogene and its normal homologue did not reveal biochemical differences that helped to explain the malignant properties of this oncogene. Finally, we report preliminary results indicating that the T24 bladder carcimoma oncogene is highly related to the transforming gene of BALB-MSV, an acute transforming retrovirus.     

甲醛致大鼠鼻腔癌与K-ras癌基因活化相关

为探讨甲醛致大鼠鼻腔癌的分子机制,对甲醛诱发的大鼠鼻腔癌细胞系FAT7中的转化序列进行检测.采用的实验方法,包括肿瘤细胞DNA与选择标志基因(neo)共转染、裸鼠成瘤性分析、Southern杂交、聚合酶链反应(PCR)和序列分析等.结果发现:在第二轮裸鼠肿瘤DNA中含有大鼠源性的K-ras基因序列,而无大鼠源性的H-ras、N-ras和p53基因序列.这表明甲醛诱发的大鼠鼻腔癌细胞系FAT7中所含的转化序列与H-ras、N-ras及p53基因无关,K-ras癌基因的活化可能参与甲醛致大鼠鼻腔癌.     

Detection and preliminary characteristics of the transforming gene (oncogene) of Ha-ras type in human stomach cancer

High molecular weight DNA prepared from three undifferentiated human stomach carcinomas was assayed for transforming activity by transfection of mouse NIH 3T3 cells. One tumor DNA sample (stomach carcinoma CaVSt) induced (the transfection efficiency: 0.02 transformants/micrograms DNA X 10(-6) cells) transformation of NIH 3T3 recipient cells. Transforming gene of Ha-ras type was identified in transformants derived from this human carcinoma. The genetic lesion responsible for the activation of the CaVSt Ha-ras oncogene is not localized in the 12-th codon for p21c-Ha-ras protein.     

HER-2/neu oncogene expression and DNA ploidy in normal human kidney and renal cell carcinoma.

Using flow cytometry (FCM), we have investigated both the DNA content (stained with propidium iodide) and HER-2/neu oncogene expression (revealed by means of an anti-HER-2/neu monoclonal antibody) in neoplastic and non-neoplastic kidney samples from 20 patients with renal cell carcinoma. All the non-neoplastic samples and 15/20 (75%) renal cell cancers showed diploid modal DNA content while the remaining 5 neoplastic sample (25%) showed both diploid and hyperdiploid cell populations. In normal kidney the level of HER-2/neu oncoprotein was low (median fluorescence values in arbitrary units = 7.5 AU, range: 4-10 AU). In diploid renal cancers the level of HER-2/neu was slightly increased (median fluorescence values = 20 AU, range: 9.5-30 AU) (p < .005). The relationship of HER-2/neu expression to the cell cycle in these tumor samples is not clear since most of the cells express the antigen in all phases of the cell cycle. On the other hand, there is an association between HER-2/neu expression and abnormal DNA content suggesting that aneuploid pattern may be biologically related to overexpression of the HER-2/neu gene.     

Transforming activity and the oncogenicity of simian adenovirus SA7 DNA]

The transforming and oncogenous activity of uncleaved DNA of simian adenovirus SA7 (AdSA7) and the products of its restriction by endonucleases R. Bam HI and R. SalI was studied. It was shown that uncleaved virus DNA transformed the rat kidney cells and rat embryo fibroblasts and induced tumors in newborn hamsters. AdSA7 DNA, hydrolysed by R. Bam HI, posessed the transforming activity. The mixture of DNA fragments, obtained after hydrolysis by R. SalI was oncogenous in hamsters.     

Mutagenic effect of the SV40 oncogene: induction of resistance to 6-mercaptopurine and serum independence

The mutagenic and transforming activity of SV40 DNA fragment, corresponding to its oncogene (the gene for large T antigen) was studied in Chinese hamster cells. After expression time of 3 to 4 days, the oncogene induced mutations of resistance to 6-mercaptopurine (6MP), while the DNA encoding the SV40 late genes, as well as DNA of Chinese hamster cells, were devoid of mutagenic activity. The value of induction ranged from 10(-4) to 10(-5). After the same expression time, the oncogene induced a typical character of oncogenic transformation - independence of serum growth factors (ser+). The value of induction of ser+ variants was somewhat higher than for resistance mutations. The study of 12 clones induced by the oncogene has shown the ser+ character to be hereditary, the expression of viral oncogene being not necessary for its maintenance. The data obtained support the hypothesis in favour of the participation of mutations of cellular genes in viral carcinogenesis.     

Isolation of a transforming sequence from a human bladder carcinoma cell line

We have isolated the component of human bladder carcinoma cell DNA that is able to transform mouse fibroblasts. The oncogenic sequence was isolated initially from a lambda phage genomic library made from DNA of a transfected mouse cell carrying the human oncogene. A subcloned insert of 6.6 kb that carried transforming activity was amplified in the plasmid vector pBR322. The subcloned oncogene has been used as a sequence probe in Southern blot analyses. The oncogene appears to derive from sequences present in normal cellular DNA. Structural analysis has failed so far to reveal differences between the oncogene and its normal cellular homolog. The oncogene is unrelated to transforming sequences detected in a variety of other types of human tumor cell lines derived from colonic and lung carcinoma and from neuroblastoma. In contrast, the EJ bladder oncogene appears closely related to one that is active in the human T24 bladder carcinoma cell line. The oncogene appears to have undergone little, if any, amplification in several bladder carcinoma cell lines.     

Sodium butyrate suppresses the transforming activity of an activated N-ras oncogene in human colon carcinoma cells

The transforming activity of DNA from a newly established undifferentiated human colon carcinoma cell line (MIP-101) was tested in the NIH-3T3 transfection assay. Southern blot analysis of the transfectant DNA revealed the presence of a human N-ras oncogene. Treatment of MIP-101 cells with the maturational agent sodium butyrate induced a more normal phenotype, including diminished growth rate, elimination of anchorage independent growth, and decreased tumorigenicity (R. Niles, S. Wilhelm, P. Thomas, and N. Zamcheck (1988) J. Cancer Invest. 6, 39). Here we report that there is a significant reduction in the transforming efficiency of the DNA from butyrate-treated MIP-101 cells. A nonspecific reduction in total DNA uptake as an explanation for these findings was eliminated by showing that there was similar uptake and expression of the thymidine kinase gene from the DNA of butyrate-treated and control MIP cells. Butyrate treatment had no detectable effect on the overall structure, methylation, and level of expression of the human N-ras gene from MIP-101 cells. An NIH-3T3 transformant ability after treatment with sodium butyrate. Although butyrate suppressed several transformed properties similar to MIP-101 cells, DNA from control and treated cultures had an identical level of transforming activity. The results suggest that the environment of the MIP cells may contain additional elements not present in the NIH-3T3 transformants which are required to observe the effect of butyrate on reduction of transforming activity.     

Activation of ras and myc proto-oncogenes in human breast carcinoma and neuroblastoma

DNA from human breast carcinoma (SK-BR-3) and neuroblastoma (LA-N-1) cell lines are capable of inducing foci of transformed NIH 3T3 cells after DNA-mediated gene transfer. The blot hybridization analysis of DNA from primary and secondary NIH 3T3 transformants identified additional sequences homologous to the c-Ha-ras 1 oncogene, and revealed amplification of nucleotide sequences homologous to the v-myc oncogene. Restriction fragments of the amplified myc-related sequences correspond to c-myc (SK-BR-3) and N-myc (LA-N-1) loci of the human genome. The results show that active Ha-ras oncogenes can coexist with altered myc oncogenes in breast carcinomas and neuroblastomas. This suggests that a multi-step mechanism involves both ras and myc genes and their cooperation in the development of these tumors.     

Activation of c-myc and c-K-ras oncogenes in primary rat tumors induced by ionizing radiation.

An activated K-ras oncogene was detected by transfection in NIH 3T3 cells and by Southern blot analysis in 6 of 12 rat skin tumors induced by ionizing radiation. The DNA from 10 of the 12 tumors also showed c-myc gene amplification and restriction polymorphisms. Evidence for tissue specificity was observed in patterns of oncogene activation, with each of three clear cell carcinomas exhibiting activation of both c-myc and K-ras oncogenes.     

Molecular cloning of the intronless EJ ras oncogene using a murine retrovirus shuttle vector

We have inserted a genomic clone of the human EJ bladder oncogene into a murine retrovirus shuttle vector. Cotransfection of this shuttle vector DNA containing the activated ras oncogene with molecularly cloned Moloney murine leukemia virus into NIH/3T3 cells was able to rescue a replicating and transforming retrovirus. The viral DNA from infected cells was excised by fusion to mouse COP-5 cells and recloned into Escherichia coli as plasmid DNA. Analysis of the recloned plasmids by size, restriction enzyme mapping, and DNA sequence indicated that approximately 5% of the recloned plasmids contained the intronless EJ ras oncogene.     

Developmental and molecular aspects of nephroblastomas induced by avian myeloblastosis-associated virus 2-O.

Avian myeloblastosis-associated virus-induced nephroblastomas are tumors consisting mainly of mesenchymal and epithelial renal elements with variable degrees of differentiation. The spatial distribution of developmental stages reflects a gradient of differentiation from less differential structures in the periphery towards more differentiated structures in the center of the lobules formed in the nephroblastomas. These heterogenic tumors contain discrete virus-cell DNA junction fragments and are therefore clonal outgrowths of a single transformed cell. These findings support the hypothesis that a mesenchymal, nephrogenic cell residual in the postembryonic kidney is the origin of the tumor, which grows by proliferation and differentiation of this target cell. All the tumors expressed higher levels of viral genomic and env messages than nontransformed tissue from the same kidney. A screening of oncogene expression with 13 different oncogenes revealed enhanced myc levels. There was, however, no rearrangement of c-myc or of the other oncogenes detected with EcoRI-digested tumor DNAs. This suggests that there is no insertion of viral elements adjacent to a c-myc. The levels of myc expression in embryonic kidneys were as high as in the tumors. Therefore, the enhanced myc expression in nephroblastomas is a reflection of the embryonic status of the tumor rather than a newly acquired function. This finding, plus the similarity of development and morphology of nephroblastomas and embryonic kidneys, suggests that the tumors arise as a result of a deficiency in a function which turns the embryonic status off.