We propose that triplex formation by the promoters of the proliferation-associated genes dihydrofolate reductase (dhfr), cyclin D1, and E2F-1 represents a physiologic mechanism for regulation of gene expression, as well as a viable strategy for possible sequence- specific antiproliferative therapies. We now have evidence that minor dhfr transcripts, initiating from the upstream (about 400) minor promoter are capable of sequence-specific triplex interaction with the dhfr core promoter. We have also identified naturally occurring heterologous transcripts which contain sequences potentially capable of triplex interaction at these promoters. We propose to develop constructs capable of directing the intracellular biosynthesis of natural or selected high affinity triplex-forming transcript sequences, to test the hypothesis that effective triplex formation by one or a synergistic combination of these promoters will interfere with gene expression in a sequence specific manner, and inhibit the abnormal proliferation of cancer cells.
The specific Aims are: 1. To evaluate the potential of minor upstream transcripts to regulate dhfr expression and cellular proliferation through intermolecular triplex formation. 2. To clone and characterize heterologous genes encoding naturally occurring triplex-forming transcripts (tft's) targeted to the human dhfr, cyclin D1, or E2F-1 promoters; and to determine the role of these tft's in physiological regulation of gene expression and cellular proliferation, in both normal and malignant cells. 3. To select (in vitro) and characterize high affinity triplex-forming RNA sequences, representing potential therapeutic modulators of gene expression, for the dhfr, cyclin D1, or E2F-1 targets. 4. To develop constructs which express the natural regulatory or selected high affinity tft sequences, and test the target-specific and potential synergistic/antiproliferative effect of these tft's expressed inside cancer cells.
