THE DIFFERENTIAL EXPRESSION OF INSULIN-LIKE GROWTH FACTOR mRNA IN TWO RAT TUMOR CELL LINES

Buffalo Rat Liver 3A (BRL) and 18, 54-SF cells are two rat tumor cell lines which have been maintained continuously in serum-free media. Both cell lines secrete rat IGF-II (>50ng/ml in three day conditioned media) and have no detectable rIGF-I peptide by RIA. The experimental design was to: 1) demonstrate that lack of rIGF-I production was due to absent transcription of rIGF-I mRNA, and 2) to compare BRL, 18, 54-SF and rat liver IGF-II RNA sizes and abundance patterns on Northern blots. RNA was prepared by the guainidine thiocyanate/LiCI technique. Polyadenylated RNA (A+) was separated from non-polyadenylated RNA (A−) by oligo dT cellulose chromatography, sized on formaldehyde-agarose gels and transferred to nylon membranes. IGF RNA sequences were detected either by hybridization to in vitro transcribed cRNA specific to each strand, or to in vitro hexamer primed DNA at a specific activity > 109 cpm/μg. Probes were to i) the carboxyl terminal E peptides and 3′ nontranslated regions which are highly specific for each of the two IGF species and ii) the 5′ region of IGF-I thus allowing detection of both IGF-I splicing variants. Non-specific binding was reduced by final washing in 0.1x SSC at 62C. IGF-I mRNA could not be detected in BRL nor 18, 54-SF cells but was easily detected in adult rat liver as 1800 and 660 base major species with ~6 intermediate forms. IGF-II mRNA was detected by the 3′ cDNA and 3′ anti-sense cRNA probes; no species were found to hybridize with the 3′ sense cRNA, indicating that all mRNAs are in the usual 5′ to 3′ sense orientation. The BRL and 18, 54-SF cells had multiple IGF-II forms (>10kb, 7.0kb, 5.8kb, 4.4kb, 3.5kb, 3.0kb, 2.7kb, 2.5kb, 1.8kb and 1.1kb). The most abundant form in BRL cells was 5.8kb in length and polyadenylated. The predominant form in 18,54-SF cells was 4.4kb in length and this was the most abundant A+ form; additionally an A- form at 1.1kb was observed. Adult rat liver had no detectable IGF-II RNA sequences. These data demonstrate that regulated expression occurs at the level of transcription. The presence of rIGF-I mRNA and peptide in the absence of rIGF-I mRNA and peptide, coupled with the ability to grow in serum free defined media support IGF-II as being a critical growth factor in these cell lines.