DECREASING THE LEVEL OF TRANSLATION INITIATION FACTOR-4E WITH ANTISENSE RNA CAUSES REVERSAL OF RAS-MEDIATED TRANSFORMATION AND TUMORIGENESIS OF CLONED RAT EMBRYO FIBROBLASTS

Transformation of cloned rat embryo fibroblasts (CREF) with the T24-ra s oncogene results in loss of contact inhibition, growth in soft agar and tumor formation in nude mice. Previously we showed that in such ce lls (CREF T24), the phosphorylation rate of protein synthesis initiati on factor 4E (eI-4E) is increased, correlating with an increase in the general rate of protein synthesis. In the present study, we have expr essed antisense RNA complementary to eIF-4E mRNA in CREF T24 cells usi ng a stably integrated vector. Cells expressing antisense RNA (CREF T2 4/AS) contained 30-50% of the normal level of eIF-4E and exhibited man y of the properties of untransformed cells. CREF T24 had a spindle-sha ped, refractile appearance, whereas CREF T24/AS grew in ordered, paral lel patterns and exhibited contact inhibition similar to untransformed CREF. The rates of growth and protein synthesis in CREF T24/AS were d ecreased compared to CREF T24 but were not as low as in CREF. The effi ciency of growth in soft agar was 11-fold lower for CREF T24/AS compar ed with CREF T24. The latency period for tumor formation in nude mice was increased from 8 days for CREF T24 to 17-27 days for CREF T24/AS a nd various clonal lines derived from them. Cell lines established from these CREF T24/AS-derived tumors were shown to have partially regaine d the eIF-4E levels characteristic of CREF T24. These results demonstr ate that many of the phenotypic alterations associated with ras-induce d malignant transformation can be reversed by a moderate reduction of the translational initiation capacity and therefore may be mediated th rough a translational mechanism. (C) 1993 Wiley-Liss, Inc.