INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS IN R3230AC MAMMARY-TUMORSOF INTACT AND DIABETIC RATS

The insulin-responsive R3230AC mammary tumor possesses type I and type II insulin-like growth factor (IGF) receptors, and membrane preparati ons display affinity cross-linking of I-125-labeled IGF-I to IGF-bindi ng proteins (IGFBPs). To identify the IGFBPs produced, Northern blotti ng analysis of poly(A)+ RNA extracts from tumor tissue was performed. Although transcripts of IGFBP-1 were not detected, intense bands were obtained at 1.7 and 6 kilobases (kb) when hybridized with radiolabeled IGFBP-2 and IGFBP-5 cDNA probes, respectively. A 2.6-kb band and a 2. 4-kb weaker band were observed after hybridization with IGFBP-3 and IG FBP-4 probes, respectively. When IGFBP-6 cDNA was used, two bands were seen: a higher mol wt band at 6.3 kb and a smaller one at 1.3 kb. Tum ors from streptozotocin-induced diabetic rats displayed an increase in the expression of IGFBP-2, and insulin treatment for 3 days normalize d the IGFBP-2 mRNA levels. Tumors from diabetic rats displayed no chan ge in IGFBP-3, -4, -5, and -6 mRNA levels from tumors of normoglycemic rats. However, tumors from insulin-treated rats showed significantly higher levels of mRNA for IGFBP-4 and IGFBP-5 than tumors from normogl ycemic or diabetic rats. A similar, but less pronounced, pattern of ch anges in IGFBP-3 mRNA was seen, whereas levels of IGFBP-6 mRNA were un changed throughout. To identify the cell type producing the mRNAs for these IGFBPs, in situ hybridization of tissue sections was used. Proce dures were established that localized three of the five IGFBPs express ed in this tumor tissue. This technique showed that IGFBP-3 mRNA trans cripts were observed mainly in endothelial cells of tumor vasculature, although they were also detected in stromal cells, IGFBP-4 was presen t mainly in tumor Btroma cells, and IGFBP-5 mRNA was expressed predomi nantly in the epithelial cells of this tumor. Expression of IGFBP-5 mR NA transcripts was significantly diminished in primary and long term c ultured R3230AC cells grown in a-Minimum Essential Medium and 10% feta l bovine serum. Tumors arising from injection of long term cultured ce lls that were injected into isologous rats contained high amounts of m RNA transcripts for IGFBP-5, suggesting the presence, in vivo, of posi tive regulators for the expression of this BP. Tumor cells cultured in the presence of insulin displayed a 2.2- to 2.5-fold increase in the expression of IGFBP-5. These findings imply a role for insulin as a re gulator of the expression of IGFBP-5 in the R3230AC adenocarcinoma.