Frequent provirus insertional mutagenesis of Notch1 in thymomas of MMTVD/myc transgenic mice suggests a collaboration of c-myc and Notch1 for oncogenesis.

Abstract

The MMTVD/myc transgenic mice spontaneously develop oligoclonal CD4+CD8 + T-ceU tumors. We used provirus insertional mutagenesis in these mice to identify putative collaborators of c-myc. We found that Notchl was mutated in a high proportion (52%) of these tumors. Proviruses were inserted upstream of the exon coding for the transmembrane domain and in both transcriptional orientations. These mutations led to high expression of truncated Notchl RNAs and proteins (86-110 kD). In addition, many Notchl-rearranged tumors showed elevated levels of full-length Notchl transcripts, whereas nearly all showed increased levels of full-length (330-kD) or close to full-length (280-kD) Notchl proteins. The 5' end of the truncated RNAs were determined for some tumors by use of RT-PCR and 5' RACE techniques. Depending on the orientation of the proviruses, viral LTR or cryptic promoters appeared to be utilized, and coding potential began in most cases in the transmembrane domain. Pulse-chase experiments revealed that the 330-kD Notchl proteins were processed into 110-and 280-kD cleavage products. These results suggest that Notchl can be a frequent collaborator of c-myc for oncogenesis. Furthermore, our data indicate that Notchl alleles mutated by provirus insertion can lead to increased expression of truncated and full-length (330/280-kD) Notchl proteins, both being produced in a cleaved and uncleaved form.

A full-length Notch1 allele is dispensable for transformation associated with a provirally activated truncated Notch1 allele in Moloney MuLV-infected MMTVD/myc transgenic mice

“…All infected Tg mice analysed (n=47) developed oligoclonal thymomas. All analysed thymomas from both genotypes (except 2 Tg +/7 /Notch1 +/+ and 1 Tg +/7 /Notch1 +/7 mice) disseminated to the spleen, lymph nodes and sometimes in other organs (kidneys or the liver), in accordance to what was previously described (Girard et al, 1996). The mean latency of tumor appearance was 65 days in the Tg +/7 / Notch1 +/+ mice (n=21) and 67 days in the Tg +/7 / Notch1 +/7 mice (n=26) ( Table 1).…”

“…We previously reported that the Notch1 gene was rearranged in more than 50% of T-cell lymphomas induced by Moloney MuLV infection of MMTV D /myc transgenic (Tg) mice (Girard et al, 1996). In these tumors, the proviruses were inserted upstream of exons coding for the transmembrane domain of Notch1 and were found in both transcriptional orientations.…”

“…Interestingly, in several tumors with a Notch1 rearrangement, overexpression of full-length Notch1 RNA (8/10 kb) and/or proteins (280/330 kD) could also be detected (Girard et al, 1996), suggesting that this event could also participate in the transformation process. We have now tested this hypothesis directly using a genetic approach.…”

“…We have now tested this hypothesis directly using a genetic approach. We used mutant mice generated by homologous recombination and which harbor a deletion of the Notch1 gene (Notch1 +/7 ) in the same region previously found to be the site of provirus integrations detected with probe D (Girard et al, 1996;Conlon et al, 1995). In these heterozygote Notch1 +/7 mice, any provirus insertion detected with probe D will necessarily occur on the normal Notch1 allele.…”

The Notch signaling pathway: Transcriptional regulation at Notch target genes

“…Notch mutations appear to collaborate with a diverse collection of other proteins dysregulated in T-ALL. Retroviral oncogenesis in mice indicates a synergistic interaction between Notch1 and c-myc [162], E2A/ pbx [163] and dominant negative forms of Ikaros [164] to enhance development of T-ALL. Apart from Notch1, Notch3 was consistently expressed in human T-ALL and dramatically reduced in clinical remission [165].…”