DIFFERENTIAL EXPRESSION OF NON-ALLELIC INSULIN GENES IN RODENT ISLET TUMOR-CELLS

We have compared the expression patterns of the non-allelic insulin 1 and 2 genes during prolonged in-vitro culture of the mouse islet cell line beta-TC3, where transformation by the SV40 T oncoprotein is targe ted to the differentiated beta-cell phenotype, and the rat islet cell line NHI-6F, in which the beta-cell phenotype is induced by transient in-vivo passage. The NHI-6F clone carries, in addition, a single copy of a transfected silent human insulin gene which contains 3 kb of regu latory sequences known to confer beta-cell-specific expression. Insuli n gene expression was measured by an assay based on a reverse transcri ption-polymerase chain reaction, to determine whether the ancestral ro dent insulin 2 genes (and the human homologue in the NHI-6F cells) are regulated differently from the duplicated rat and mouse insulin 1 gen es. We have shown that activation of insulin gene expression in the NH I-6F cells includes transcriptional activation of all three genes, but that extended propagation of tumour cells in vitro leads to a selecti ve and equal decline in the quantities of transcripts from the rat 2 a nd human genes relative to transcripts from the rat 1 gene. In the lat er passages, insulin transcripts were derived almost exclusively from the rat 1 gene. In early in-vitro passages of the mouse endocrine cell line beta-TC3, the expression pattern of the mouse 1 and 2 insulin ge nes resembled that seen in isolated mouse islets. After more than 45 i n-vitro passages, expression of the duplicated mouse 1 gene decreased tenfold when compared with the ancestral mouse 2 gene. As previously s hown for NHI-6F cells, the differential expression of non-allelic insu lin genes in the beta-TC3 line was also clearly evident at the cellula r level, where a subpopulation of cells selectively expressed readily detectable levels of mouse C-peptide 2 immunoreactivity while devoid o f C-peptide 1. Our results suggest that the maintenance of insulin gen e expression in rodent tumour cells is influenced by enhancer sequence s which are not shared by the ancestral and duplicated insulin genes, and that either species-specific conditions or transformation-related differences exist between the rat and mouse cell lines that govern whi ch gene remains active during prolonged in-vitro propagation.