Integration of viral sequences into the c-myc gene in two mammary adenocarcinomas induced by polyomavirus in athymic nude mice.

We report the analysis of polyomavirus (Py) DNA integration into chromosomal DNA of two Py-induced mammary adenocarcinomas of athymic nude mice. Prior observations had established that these tumors had high levels of episomal Py DNA, making analysis of integration sites difficult. Propagation of tumor cells in culture allows the isolation of lines which have lost episomal Py DNA but are still tumorigenic and thus can be used for in situ and Southern analysis of Py sequences. The data reported here support the conclusion that Py DNA integrated into and next to the c-myc gene, adding further importance to this tumor system which, in its modifications of c-myc expression, appears to be similar to some human mammary cancers. In situ hybridization experiments on metaphase chromosomes of tumor cells showed that (i) in both cases, there was a single integration site at the same position on the same chromosome in all cells of a given tumor, and (ii) integration sites were different in the two tumors; in one, it was located on chromosome 15, near the c-myc proto-oncogene, and in the other, it was situated in the distal part of chromosome 1. We have demonstrated a probable rearrangement between chromosome 1 and chromosome 15, in the region of Py insertion, thus suggesting that a specific site on chromosome 15 is involved in tumorigenesis. The discovery that Py DNA was integrated at specific sites in host chromosomes raised the questions of whether such integrations were correlated with the activation of specific oncogenes. The rearrangements of the c-myc proto-oncogene observed on Southern blot analysis for both tumors, along with similar integration patterns of Py sequences, the overexpression of the c-myc gene, and the synthesis of abnormal oversized hybrid transcripts between c-myc and Py genes, favor this hypothesis. Finally, the analysis of episomal Py DNA in various tumors shows viral populations presenting a specific deletion in a part of the Py late region. This deleted region in the episomal virus genome was systematically found integrated in chromosomal DNA, thus arguing for the importance of Py integration in the induction of mammary tumor.

ras oncogene activates the intracisternal A particle long terminal repeat promoter through a c-AMP response element.

To understand the mechanism responsible for the high expression of Intracisternal A Particles (IAP) in murine cells transformed by the Kirsten Mouse Sarcoma Virus (Ki-MSV), we have investigated the effect of p21ras on IAP transcription. By transient cotransfections of IAP LTR-CAT plasmids with v-Ki-ras and c-Ki-ras expression vectors, we have found that p21v-Ki-ras, and the p21c-Ki-ras to a lesser extent, stimulate the promoter activity of the 5' IAP LTR. We constructed several plasmids containing the CAT gene under control of IAP LTRs deleted in different regions. CAT assays demonstrate that the ras responsive sequence is a 16 bp oligonucleotide containing an effective c-AMP Response Element (CRE) TGACGTCA, which is located in the U3 region, 20 bp upstream of the CAAT box.

Paradoxical effects of recombinant mouse beta-interferon on different expression levels of intracisternal A particle genes.

We have studied the effect of mouse recombinant beta-Interferon on the expression of the intracisternal A particle (IAP) genes in a transformed mouse fibroblast cell line. Northern and immunoprecipitation analysis showed an increase in specific transcripts and polypeptides. Run-on experiments on isolated nuclei and transfection assays with an IAP long terminal repeat construct, coupled with the chloramphenicol acetyl transferase gene, showed no enhancement of the transcription rate following beta-IFN treatment. This suggests that the increase in IAP expression depends on a post-transcriptional event. Electron microscopic analysis revealed that in spite of the higher levels of IAP related polypeptides, the number of particles was significantly reduced by interferon.

Dexamethasone stimulates expression of transposable type A intracisternal retroviruslike genes in mouse (Mus musculus) cells.

Dexamethasone treatment enhances expression of transposable intracisternal type A particles (IAP) at RNA and proteins levels in a murine retrovirus-transformed cell line (Ki-BALB). This effect was ascertained by electron microscopic numeration of IAP. By sequence comparison, we located glucocorticoid-responsive elements in IAP long terminal repeats. Their regulatory potential was tested on the promoter activity of an IAP long terminal repeat construct coupled with the chloramphenicol acetyltransferase gene. Our findings suggest that the IAP activation by dexamethasone occurs at the level of transcription.

Differential expression of c-myc and c-fos oncogenes during the differentiation of human leukemic cells by Ara-C.

1-B-D-arabinofuranosyl cytosine (Ara-C) is, at very low concentrations, an inducer of monocytic differentiation of human leukemic cells. By what mechanisms this differentiation is obtained and how the monocytic pathway is determined, is not yet understood. We studied the effect of Ara-C on the RNA transcript levels of two c-oncogenes often associated to cell proliferation and differentiation. In the monoblastic U-937 cell line, where Ara-C has a maximum (100%) monocytic differentiation, RNA transcript level of c-myc is not detectable, whereas c-fos levels are rapidly increased. On the other hand, in the bipotential promyelocytic HL-60 cells, Ara-C induces only 40% monocytic differentiation after 7 days; this is associated to a weaker decrement of c-myc expression; c-fos is however greatly induced 3 log after 2 days treatment, but before monocytic phenotype is observed. Ara-C, at low concentrations, may, by arresting cell proliferation, restore the leukemic's cell proliferation/differentiation equilibrium. The rapid inducement of c-fos expression may allow to foresee a rapid prescreening of Ara-C sensitive-blasts.

Organization and state of methylation of endogenous type C retroviral sequences in 129 mouse differentiated and undifferentiated teratocarcinoma cell lines.

Attempts to activate type C endogenous viruses in 129 mouse fibroblasts and in teratocarcinoma-derived cell lines have never been successful, although the genome of these cells contains xenotropic virus-related sequences. We have investigated the arrangement of these sequences and their methylation state by DNA restriction endonuclease digestion, electrophoresis of digests in agarose gels, Southern blotting and hybridization with specific probes. Our results show that the majority of the sequences are organized into two complete provirus families integrated at multiple sites in the cell genome and that they are hypermethylated in embryonal carcinoma cells as compared with differentiated cells. Having previously found a higher expression of viral RNA in 129 derived embryonal carcinoma cells, our data indicate an apparent direct correlation between methylation and type C virogenes expression.

Interferon-mediated regulation of myc and Ki-ras oncogene expression in long-term-treated murine viral transformed cells.

Long-term treatment of a murine retroviral-transformed cell line (Ki-Balb) with 50 units/ml of interferon (IFN) resulted in a morphological reversion. The effects of IFN on myc and Ki-ras oncogene expression were examined after 6 months of treatment. mRNA dot and Northern blots hybridization analysis reveal that the expression of c-myc at the RNA level decreases by about fourfold. This reduction in the c-myc mRNA appears to be selective since in the same cells v-Ki-ras and an endogenous retroviral gene, intracisternal A particles (IAP), are increased four- and threefold, respectively. No significant inhibition of cellular growth and cell-cycle distribution was observed in IFN-Ki-Balb-treated cells.

Biochemical characterization of endogenous type C virus information in differentiated and undifferentiated murine teratocarcinoma-derived cell lines.

Undifferentiated teratocarcinoma cells express sixfold-higher levels of endogenous xenotropic type C virus-related RNA than differentiated cells. Three species of polyadenylated viral RNA (35S, 24S, and 14S) have been identified in the undifferentiated teratocarcinoma cells. Paradoxically, neither viral particles nor viral proteins have been detected in these cells.