The important role of activated K-ras oncogene expression in the development of non-small cell lung cancer and its strong association with relapse and significantly decreased survival is clear. The overall goal of this proposal is to investigate a new strategy in the design of specific anti-K-ras transcriptional inhibitors which may offer a more effective treatment to reduce the significant mortality associated with non-small cell lung cancer. We have developed stable nuclease resistant oligonucleotides which form triplex DNA in a sequence specific manner and inhibits nuclear protein binding in the K-ras promoter. We now propose to study the ability of these human and murine specific triplex forming oligonucleotides (TFOs) to bind to specific DNA sequences in the K-ras promoter in intact NSCLC cells, to repress transcription of activated K- ras in a gene specific manner both in vitro and in vivo, and to inhibit lung tumor growth in animal models.
The specific aims of this proposal are: 1. To determine the effect of triplex formation by the K-ras promoter targeted phosphorothioate (PS) oligonucleotides on nuclear protein binding and transcription in vitro by the K-ray promoter. 2. To determine ability of K-ras PS-oligonucleotides to bind to their target sequence in the K-ras promoter in intact human non-small cell lung cancer (NSCLC) cells and characterize its effect on K-ras transcription and gene expression, cell proliferation, and cell viability in both NSCLC and normal lung cells. 3. To evaluate the effect of K-ras targeted triplex forming PS- oligonucleotides on K-ras transcription and expression as well as tumor growth characteristics in human NSC lung tumors in nu/nu mice. 4. To assess the ability of K-ras PS-TFOs to repress human K-ras promoter activity in transgenic mice. 5. To determine the ability of murine specific K-ras PS-triplex forming oligonucleotides to repress """"""""whole animal"""""""" K-ras transcription/expression and the potential subsequent pharmacological effects both in mice bearing primary lung tumors and in normal mice. The accomplishment of these specific aims will allow the identification of potential transcriptional inhibitors of K-ras in vivo. The analysis of epidermal growth factor receptor (EGF-R) and insulin receptor (I-R) genes whose promoters contain similar protein binding sequences necessary for their transcription, will further allow us to critically evaluate the sequence and functional specificity of these K-ras targeted compounds in vivo. Development of both human and murine specific K-ras phosphorothioates will afford us the unique ability here to determine the potential for triplex forming oligonucleotides to inhibit both human transgenic and native murine gene expression and to determine the resulting pharmacological effects in whole animals. These studies will provide critical information on the potential for antigene therapy in humans.
| Ebbinghaus, S W; Vigneswaran, N; Miller, C R et al. (1996) Efficient delivery of triplex forming oligonucleotides to tumor cells by adenovirus-polylysine complexes. Gene Ther 3:287-97 |
| Vigneswaran, N; Mayfield, C A; Rodu, B et al. (1996) Influence of GC and AT specific DNA minor groove binding drugs on intermolecular triplex formation in the human c-Ki-ras promoter. Biochemistry 35:1106-14 |
