Decreased virus population diversity in p53-null mice infected with weakly oncogenic Abelson virus

The Abelson murine leukemia virus (Ab-MLV), like other retroviruses that contain v-onc genes, arose following a recombination event between a replicating retrovirus and a cellular oncogene. Although experimentally validated models have been presented to address the mechanism by which oncogene capture occurs, very little is known about the events that influence emerging viruses following the recombination event that incorporates the cellular sequences. One feature that may play a role is the genetic makeup of the host in which the virus arises; a number of host genes, including oncogenes and tumor suppressor genes, have been shown to affect the pathogenesis of many murine leukemia viruses. To examine how a host gene might affect an emerging v-onc gene-containing retrovirus, we studied the weakly oncogenic Ab-MLV-P90A strain, a mutant that generates highly oncogenic variants in vivo, and compared the viral populations in normal mice and mice lacking the p53 tumor suppressor gene. While variants arose in both p53+/+ and p53-/- tumors, the samples from the wild-type animals contained a more diverse virus population. Differences in virus population diversity were not observed when wild-type and null animals were infected with a highly oncogenic wild-type strain of Ab-MLV. These results indicate that p53, and presumably other host genes, affects the selective forces that operate on virus populations in vivo and likely influences the evolution of oncogenic retroviruses such as Ab-MLV.     

Analysis of polyadenylation site usage of the c-myc oncogene.

The c-myc gene contains 2 well conserved polyadenylation (pA) sites. In all human and rat cell lines from various differentiation stages and tissue types the amount of mRNA terminating at the second pA site is 6-fold higher than the amount ending at the upstream site. This is not due to a difference in stability of the two mRNA types and therefore must be due to preferential usage of the downstream site. The usage of the pA sites is not altered during growth factor induction of quiescent cells. We have not been able to detect differences in behavior between mRNAs ending at either pA site. Both types of mRNA are induced upon treatment of cells with cycloheximide. Furthermore, we have shown that the poly(A) tail of c-myc mRNA is lost during degradation of the messenger, as was described previously for c-myc mRNA in an in vitro system. The time required for the loss of the poly(A) tail is similar to the half-life of c-myc mRNA.     

Induction of clonal monocyte-macrophage tumors in vivo by a mouse c-myc retrovirus: rearrangement of the CSF-1 gene as a secondary transforming event.

A mouse retrovirus containing the c-myc oncogene was found to induce tumors of mononuclear phagocytic cells in vivo. All tumors expressed the c-myc retroviral gene but not the endogenous c-myc gene (with one exception), and virtually all tumors were clonal with a unique proviral integration. This observation, coupled with a lag time in tumor formation, suggests that a second event, in addition to c-myc proviral integration, is necessary for the generation of neoplastic cells in vivo. All of the tumor cells expressed high levels of mRNA for both the putative colony-stimulating factor 1 (CSF-1) receptor (c-fms proto-oncogene product), as well as the c-fos proto-oncogene. Although all of the tumor cells proliferated in culture without the addition of exogenous CSF-1, which is required for the proliferation of primary macrophages partially transformed by the same c-myc retrovirus, several phenotypes were observed with respect to the expression of CSF-1 and granulocyte-macrophage CSF and to their growth factor responsiveness. The proliferation of one tumor, which secreted high levels of CSF-1, was blocked by specific anti-CSF-1 serum. This tumor was found to express altered CSF-1 mRNA and to have a DNA rearrangement at the CSF-1 locus. In this particular case, the data indicate that a CSF-1 gene rearrangement was the secondary event in development of the tumor. The pleiotropy of phenotypes among the other tumors indicated that there are a variety of other mechanisms for such secondary events which can be investigated with this system.     

Murine T lymphomas in which the cellular myc oncogene has been activated by retroviral insertion

The myc oncogene is implicated here in T lymphocyte neoplasia. Cloning revealed a retroviral insert 0.7-1.3 kb 5' to c-myc in two T lymphomas induced by Soule murine leukemia virus and in a spontaneous T lymphoma ( Tikaut ) of an AKR mouse, a strain in which leukemogenesis involves recombinant retroviruses (MCF viruses). The tumor c-myc mRNAs appear normal but their level is approximately 5-fold higher than in most T lymphomas lacking c-myc rearrangement. Since each insert would be transcribed away from c-myc, its activation cannot involve the promoter of the long terminal repeat (LTR) but could reflect an enhancer, like that demonstrated within the Soule LTR. The Tikaut provirus has an MCF-like recombinant env gene and LTR sequence. MCF-like inserts were found near c-myc in seven of 31 other AKR T lymphomas; two lie 3' to c-myc and the five upstream are oriented away from c-myc. We conclude that a quarter of retrovirus-induced T lymphomas involve activation of c-myc, probably via the LTR enhancer.     

Transformation of quail embryo fibroblasts by a retrovirus carrying a normal human c-myc gene.

We have constructed avian retroviruses expressing the human c-myc oncogene. These viruses morphologically transformed primary quail embryo fibroblasts upon transfection and infection. Transformed cells produced viruses harboring a spliced c-myc gene and contained high levels of p64-67c-myc protein. One of these infectious viruses, vSX-AHM, was molecularly cloned and the nucleotide sequence of the spliced c-myc insert determined. No mutation was found within the c-myc coding sequence of this transforming clone when compared to the normal genomic progenitor. Thus, we concluded that no mutation within the human c-myc gene is required to induce primary avian embryo fibroblast transformation.     

Presence of papillomavirus genomes and amplification of the c-myc and C-Ha-ras oncogenes in invasive cancers of the uterine cervix

Invasive squamous cell carcinomas of the uterine cervix from 12 untreated patients were examined for the presence of human papillomavirus (HPV) genomes and for the state of the oncogenes c-myc and c-Ha-ras. Blot hybridization experiments have demonstrated the presence of the genome of HPV type 16 (HPV 16) in six tumors and that of the genomes of HPV types weakly related to HPV 16 or HPV 18 in five others. In the nine tumors corresponding to advanced stages of the disease (stages 3 and 4) there was a 3-30 fold amplification of c-myc and/or c-Ha-ras. A concomitant amplification of both oncogenes was found in eight cancers. In only one of the three tumors confined to the cervix (stage 1), the oncogene c-Ha-ras was weakly amplified. Neither HPV DNA sequences, nor oncogene amplification were detected in the leukocytes of five patients. Thus, it seems likely that specific HPV types play a role in the development of carcinomas of the uterine cervix, and that cellular oncogenes, activated through an amplification process, are involved in at least some steps of tumor progression.     

CWE纯系小鼠脑组织发育过程中细胞癌基因及其表达研究

 本文以Ha-ras.c-fos、c-myc、v-erbB、mos五种癌基因片段为分子探针,应用斑点杂交技术,对CWE纯系小鼠脑组织发育的四个时期(胚胎期、新生期、性成熟期、体成熟期)的RNA进行分子杂交分析。发现:在实验各期中这几种癌基因的表达有较明显的变化。Ha-ras、c-fos、c-myc基因的表达在胚胎期和新生期脑组织中较高;v-erbB基因的表达在性成熟期脑组织中较高;mos基因在实验各期均无表达。DNA斑点杂交结果:小鼠脑组织发育各期均未观察到癌基因扩增现象。Southern杂交结果:CWE小鼠发育的各期均显示:5.4kb、3.4kb、1.8kb、1.0kb四条c-fos基因区带;7.4kb、4.5kb、3.4kb三条c-myc基因区带。结果表明:不同细胞癌基因在小鼠胚胎发育和胚后发育中有不同的表达。     

Analysis of the structure and expression of the c-myc oncogene in cervical tumor and in cervical tumor-derived cell lines

The structure of the c-myc oncogene in 17 cervical tumors and patient-matched nontumor tissues from Chinese patients residing in Taiwan was analysed. In contrast to recent reports on Mexican patients, none of the samples showed rearrangements and sequence amplification in the c-myc gene. The discrepancy may be explained by different carcinogenesis mechanisms being in operation in different geographic regions. Although no structural alterations in the c-myc gene were found in seven cervical carcinoma cell lines analysed, Northern blot analysis indicated different levels of c-myc gene expression which may be related to the presence of human papillomavirus (HPV) sequence in the cell and suggests a possible c-myc-hpv interaction in some stages of the transformation process.     

The Parodi-Irgens feline sarcoma virus and simian sarcoma virus have homologous oncogenes, but in different contexts of the viral genomes

We have identified the oncogene and the putative transforming protein of the Parodi-Irgens feline sarcoma virus (PI-FeSV). The PI-FeSV is defective and needs a helper virus for its replication. The v-onc sequences in the PI-FeSV were found to be related to the v-sis sequences of the simian sarcoma virus (SSV). PI-FeSV nonproducer cells express two viral RNAs, a 6.8-and a 3.3-kilobase RNA. The 6.8-kilobase RNA contains gag, sis, and env sequences but lacks the pol gene. The 3.3-kilobase RNA, on the other hand, contains only env sequences. We have detected one feline leukemia virus-related protein product in these cells, namely, a 76-kilodalton protein which contains determinants of the feline leukemia virus gag proteins p15 and p30. The v-sis sequences in the PI-FeSV have been located near the 5' end of the viral genome. Taken together, these results imply that the p76 protein contains both feline leukemia virus gag and sis sequences and probably is the transforming protein of this virus. In contrast, in SSV the sis sequences are located towards the 3' end of the viral genome, and the sis protein is thought to be expressed via a subgenomic RNA. PI-FeSV and SSV therefore use different schemes to express their onc-related sequences. The v-sis sequences in the PI-FeSV contain restriction sites which reflect the different origin of the v-sis sequences in the PI-FeSV and SSV. The homologous oncogenes of the PI-FeSV and SSV thus were transduced by two different retroviruses, feline leukemia virus and the simian sarcoma-associated virus, apparently from the genomes of different species.     

Association of c-myc protein with enzymes of DNA replication in high molecular weight fractions from mammalian cells.

Oncoprotein c-myc is expressed in proliferating but not quiescent mammalian cells, and its overexpression or inappropriate expression is associated with malignant transformation. However, in spite of an intense interest, the normal function of this protein has remained elusive. As a step towards the elucidation of the function of c-myc protein, we studied its distribution within several types of cells, including HL 60, K 562, COLO 320, and CHEF/18 cells. In all of the cells studied, c-myc protein was detected in high molecular weight protein fractions, in 350-600 Kd range, in gel-exclusion chromatography and sucrose gradient centrifugation. This distribution of c-myc protein coincided with the distribution of DNA polymerase alpha and several other enzymes necessary for DNA replication. The data suggest that c-myc product may be a component of the replitase complex of enzymes involved in nuclear DNA replication.     

Analysis of the expression of the oncogene c-myc in human breast adenocarcinoma

The c-myc oncogene was characterized and its expression analyzed in 32 mammary adenocarcinomas and in 2 benign breast tumors from 34 untreated patients. Southern blot hybridization experiments have demonstrated the amplification of the oncogene (3 to 30 fold) in 3 carcinomas. The analysis of total RNA by Northern blot revealed the presence of a 2.4 kb c-myc RNA band. In 7 out of 10 carcinomas from patients with 3 or more than 3 lymph node metastases the level of c-myc expression evaluated by dot blot analysis was 4 to 14 fold greater than that of normal human tissues. In only 5 out of 22 carcinomas from patients without lymph node metastases or less than 3 invaded nodes the level of c-myc expression was also higher (4 to 10 fold). The level of c-myc expression was not significantly enhanced in the 2 benign tumors. It is suggested that the c-myc gene activation could be associated to a higher degree of malignancy of mammary carcinomas.     

Preparation and characterization of specific antisera directed against different polypeptide domains encoded by the c-myc oncogene for studying the expression of this gene introduced into quail or rat cells

By using bacterial expression vectors, we have prepared antisera directed against two polypeptidic domains encoded by exons 2 and 3 of the human c-myc oncogene. These antisera which detect specifically the human c-myc proteins allow us to analyse the expression of human c-myc gene activated by retroviral sequences and introduced in quail embryo cells (QEC) or in established rat embryo fibroblastic cell line (208 F). Although human myc mRNA are expressed in the two cell types, the p64/p67 human c-myc proteins are only detected in the QEC.     

Tetraplex DNA transitions within the human c-myc promoter detected by multivariate curve resolution of fluorescence resonance energy transfer

The nuclease hypersensitive element (NHE) III(I) of the c-myc promoter regulates the expression of oncogene c-myc and hence is an important anti-cancer target. Paranemic secondary structure formation within the promoter has been implicated in mechanistic regulation models. Here, it is shown that two monomeric tetraplexes form within the c-myc promoter, which coexist in solution. The development and application of a new experimental approach for detection of conformation transitions in nucleic acids [which exploits the sensitivity of fluorescence resonance energy transfer (FRET) for theoretical spectral resolution by multivariate curve resolution-alternating least-squares (MCR-ALS) method] has been used for this study. The pK(a) for tetraplex transitions are centered around 5.9 +/- 0.2 (between two intercalation topologies) and 6.8 +/- 0.1 (tetraplex to random coil). The presence of two tetraplexes has been further confirmed by S1 nuclease digestion. Finally, it is established that MCR-ALS analysis of FRET at different temperatures, pH, and salt concentrations allows resolution of pure species. Results are discussed in the light of recent observations implicating paranemic DNA motifs within the c-myc NHE in regulation of the oncogene. This method has several advantages over other methods vis-à-vis, high sensitivity and linear detection over a wide concentration range and, particularly, potential applications in intracellular probing.