Activity of the independently regulated human c-myc P0 promoter has been associated with the undifferentiated status of leukemia cells as well as the hormone-independent proliferation of breast cancer cells. The P0 transcript is distinguished from the predominant P1 and P2 c-myc mRNAs by an ∼639-nucleotide extension of the 5′-untranslated region. We hypothesized that this complex 5′-untranslated RNA sequence unique to the P0 transcript may contribute significantly to the composite regulation of the c-myc locus and that enforced intracellular synthesis of the isolated P0 5′-UTR, out of its native sequence context, might amplify or dominantly interfere with its normal regulatory function. Human tumor (HeLa) cells in which the isolated P0 5′-UTR was ectopically expressed displayed a dramatic decrease in anchorage-independent proliferation. Furthermore, P0 5′-UTR-expressing HeLa cells failed to form tumors when inoculated into SCID mice. This loss of tumorigenicity was associated with increases in levels of the c-Myc1 (p67) and c-Myc2 (p64) proteins and a 3- to 5-fold elevation of spontaneous apoptotic index. These results demonstrate that an isolated 5′-untranslated RNA sequence can be attributed potent in trans gene-regulatory and phenotype-altering capabilities and that extrinsic alterations in c-myc regulation can be utilized to reestablish the natural proapoptotic (tumor suppressor) activities associated with this protooncogene. © 2003 Elsevier Science (USA). All rights reserved.