Methylation similarities of two CpG sites within exon 5 of human H19 between normal tissues and testicular germ cell tumours of adolescents and adults, without correlation with allelic and total level of expression.

Ajm Gillis,Ajmh Verkerk,Jwj Oosterhuis,Lhj Looijenga,Rjhlm Van Gurp,Mc Dekker

doi:10.1038/bjc.1997.453

Abstract

Testicular germ cell tumours (TGCTs) of adolescents and adults morphologically mimic different stages of embryogenesis. Established cell lines of these cancers are used as informative models to study early development. We found that, in contrast to normal development, TGCTs show a consistent biallelic expression of imprinted genes, including H19, irrespective of histology. Methylation of particular cytosine residues of H19 correlates with inhibition of expression, which has not been studied in TGCTs thus far. We investigated the methylation status of two CpG sites within the 3' region of H19 (exon 5: positions 3321 and 3324) both in normal tissues as well as in TGCTs. To obtain quantitative data of these specific sites, the ligation-mediated polymerase chain reaction technique, instead of Southern blot analysis, was applied. The results were compared with the allelic status and the total level of expression of this gene. Additionally, the undifferentiated cells and differentiated derivatives of the TGCT-derived cell line NT2-D1 were analysed. While peripheral blood showed no H19 expression and complete methylation, a heterogeneous but consistent pattern of methylation and level of expression was found in the other normal tissues, without a correlation between the two. The separate histological entities of TGCTs resembled the pattern of their nonmalignant tissues. While the CpG sites remained completely methylated in NT2-D1, H19 expression was induced upon differentiation. These data indicate that methylation of the CpG sites within exon 5 of H19 is tissue dependent, without regulating allelic status and/or total level of expression. Of special note is the finding that, also regarding methylation of these particular sites of H19, TGCTs mimic their non-malignant counterparts, in spite of their consistent biallelic expression.

Highlights

In contrast to the finding of biallelic expression of H19, one of the imprinted genes expressed predominantly during early development (Brunkow and Tilghman, 1991; Poirier et al, 1991; Lustig et al, 1994; Leighton et al, 1995), in only a certain percentage of the different neoplasms studied, we reported on the consistent biallelic expression of H19 in human testicular gern cell tumours of adolescents and adults (TGCTs) (Van Gurp et al, 1994; Verkerk et al, 1996), which has recently been confirmed by others
RNAase protection analysis and reverse transcription polymerase chain reaction To obtain a general impression of the total level ofH19 expression in the samples included in this study, we applied RNAase protection
We have recently found a Testicular germ cell tumours (TGCTs)-derived cell line, known as NCCIT (Damjanov et al, 1993), that expressed both parental alleles of H19 upon differentiation induced by retinoic acid

Summary

MATERIALS AND METHODS

A cDNA fragment of the human H19 gene (exon 5: position 3030-3375) (Brannan et al, 1990) including the polymorphic RsaI site (position 3238) was cloned into SacIISmaI-digested PGEM3Z plasmid (Promega). The pellets were resuspended in 70 pl of water and placed on ice. After addition of 30 p1 of amplification mixture [133 mm sodium chloride, 67 mM Tris HCI pH 8.9, 17 mM magnesium sulphate, 0.03% gelatin, 670 gM dNTPs, 10 pmol of gene-specific primer HP-2 (5'-TGCTGAAGCCCTGGTGGG-3'), 10 pmol of the longest linker primer and 1 unit of Taq-polymerase (Promega)], the samples were amplified for 18 cycles consisting each of 1 min at 95°C, 2 min at 60°C, and 2.5 min at 72°C, with a 5 s extension for each cycle. Samples were placed on ice, and 5 p1 of labelling mixture [40 mm sodium chloride, 20 mM Tris HCI pH 8.9, 5 mm magnesium sulphate, 0.001% gelatin, 2 mM dNTPs, 2.5 pmol of gene-specific primer HP-3 (5'-TCGGAGCTTCCAGACTAG-3') end-labelled with T4-polynucleotide kinase (New England Biolabs) and [y32P]ATP] and 1 unit of Taq polymerase were added. RNA from the different time points was isolated and studied for H19 expression as described above

RESULTS

DISCUSSION

C Cancer Research Campaign 1997