The aim of this proposal is to determine whether maintenance of the human cancer cell tumorigenic phenotype depends on the continued presence of activated ras oncogenes. Three different experimental protocols are proposed: 1) activated ras alleles will be inactivated by targeted homologous recombination, or by the expression of a doubly mutant ras dominant negative allele; 2) dominant negative alleles of proteins in the 2 well characterized ras signalling pathways will be used to selectively disrupt each pathway; and 3) ras antisense oligonucleotides will be used to modify the in vitro growth properties and tumorigenic potential of human tumor cells containing activated ras alleles. The human tumor cells used for these experiments are heterozygous for activating mutations in H-, K- or N-ras. Assays of genetically modified or oligonucleotide-treated cells will include biochemical examination of ras and ras-dependent signalling pathways, growth and morphology in vitro, and tumor formation in mice.
|Gupta, S; Stanbridge, E J (2001) Paired human fibrosarcoma cell lines that possess or lack endogenous mutant N-ras alleles as experimental model for Ras signaling pathways. Methods Enzymol 333:290-306|
|Gupta, S; Stuffrein, S; Plattner, R et al. (2001) Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells. Mol Cell Biol 21:5846-56|
|Bernhard, E J; Stanbridge, E J; Gupta, S et al. (2000) Direct evidence for the contribution of activated N-ras and K-ras oncogenes to increased intrinsic radiation resistance in human tumor cell lines. Cancer Res 60:6597-600|
|Gupta, S; Plattner, R; Der, C J et al. (2000) Dissection of Ras-dependent signaling pathways controlling aggressive tumor growth of human fibrosarcoma cells: evidence for a potential novel pathway. Mol Cell Biol 20:9294-306|
|Plattner, R; Gupta, S; Khosravi-Far, R et al. (1999) Differential contribution of the ERK and JNK mitogen-activated protein kinase cascades to Ras transformation of HT1080 fibrosarcoma and DLD-1 colon carcinoma cells. Oncogene 18:1807-17|
|Bleuel, K; Popp, S; Fusenig, N E et al. (1999) Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc Natl Acad Sci U S A 96:2065-70|
|Cardone, M H; Roy, N; Stennicke, H R et al. (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science 282:1318-21|