Hypervariable allelic expression patterns of the imprinted IGF2 gene in tumor cells

Abstract

The IGF2 gene, which encodes a growth factor, is subject to genomic imprinting. The frequently observed loss of IGF2 imprinting in a variety of tumors has been suggested to contribute to neoplasia. Since these reports have not documented the imprinting status of IGF2 at the cellular level, it cannot be excluded that the imprinting status might vary within the tumor. The possibility that loss of IGF2 imprinting in neoplastic cells re¯ects random imprinting patterns, was therefore addressed. We show here that individual cell populations of the JEG-3 choriocarcinoma cell line display heterogenous imprinting patterns of both IGF2 and H19. In addition, a lack of correlation between IGF2 and H19 imprinting status suggests that any regional parental imprint has been functionally lost. This notion is reinforced by the observation that JEG-3 cell subclones display a range of promoter-speci®c IGF2 allele usage. Moreover, we observed that the imprinting status of H19 and IGF2 were di erentially modulated in JEG-3-derived tumors generated in nude mice. The results suggest that allele-speci®c expression of IGF2 operates in the absence of a parental imprint. Finally, our observations urge caution with respect to the general interpretation of biallelic expression as`loss of imprinting'.

Loss of imprinting in normal tissue of colorectal cancer patients with microsatellite instability

“…a, Gene-specific cDNA, derived from reverse transcription with an IGF2 downstream primer, was amplified using promoter-specific primers. PCR products were subjected to Southern allelespecific hybridization (SASH) using allele-specific oligonucleotide probes 25 . Promoters 3 and 4 showed biallelic expression in all samples examined, whereas expression from promoters 1 and 2 was not detectable.…”

“…To confirm that biallelic expression was due to LOI and not to a shift in promoter usage to P1, we did promoter-specific RT-PCR, using exon-specific primers (exon 3 for P1, exon 4 for P2, exon 5 for P3, and exon 6 for P4). The PCR products were then analyzed using allele-specific oligonucleotides as described 25 , with reconstitution controls done in parallel. In every case tested, biallelic expression in both tumor and normal specimens was observed from P3 and P4, which are both normally imprinted, and not from P1 (Fig.…”

Stochastic epigenetic variation as a driving force of development, evolutionary adaptation, and disease

“…For example, cell lines propagated clonally show a high frequency of random monoallelic expression (19). This epigenetic instability may have been first described while observing individual cancer cells (20), and data show clear epigenetic differences between identical twins (21). In evolutionary biology, social insects show environment-mediated phenotypic differences in social castes, and the distribution of those differences can be selected for (22), leading those authors to speculate that an epigenetic mechanism might be involved (23); the bee would be an outstanding model for testing these ideas.…”

The H19 Transcript Is Associated with Polysomes and May Regulate IGF2 Expression in trans

“…To ensure a linear amplification of low abundance IGF2 cDNAs in the sucrose gradient analyses, the oligo primers were 5Ј-labeled and the number of cycles were reduced to 25. This approach was verified by mixing experiments (not shown) as has been described (21). The resulting PCR products were analyzed on 8% polyacrylamide-urea sequencing gels, as has been described (21).…”

“…This approach was verified by mixing experiments (not shown) as has been described (21). The resulting PCR products were analyzed on 8% polyacrylamide-urea sequencing gels, as has been described (21).…”