C-Raf-1 protein kinase is not essential for Ras transformation of mouse embryo fibroblasts.

Transfection of primary cells with mutated oncogenic ras plus a cooperating oncogene such as myc results in the acquisition of the transformed cell phenotype. The pathways downstream of Ras that are required for transformation are an active topic of research. The Raf-MEKK-MAP kinase pathway is triggered by activation of Ras and thought to be important in Ras transformation of rodent fibroblasts. To further explore the involvement of this pathway, fibroblasts from homozygous knock out c-Raf-1 mouse embryos (20 KO) and wild-type c-Raf-1 mouse embryos (16 WT) were transfected with H-ras and myc(v). The resulting cell line derived from the knock out cells grew slower both in tissue culture and had a longer latency period as tumors than the transformed cell line from the wild-type cells. Both cell lines were however able to form tumors in nude mice. These results suggest that c-Raf-1 is not required for Ras transformation in this system

RAS-Mediated radiation resistance is not linked to MAP kinase activation in two bladder carcinoma cell lines.

The expression of activated RAS oncogenes has been shown to increase radioresistance in a number of cell lines. The pathways by which RAS leads to radioresistance, however, are unknown. RAS activates several signal transduction pathways, with the RAF-MAP2K-MAP kinase pathway perhaps the best studied. MAP kinase has also been shown to be activated by radiation through this pathway. Given the important role of MAP kinase in multiple signaling events, we asked if radioresistance induced by RAS was mediated through the activation of MAPK. Cells of two human bladder carcinoma cell lines were used, one with a mutated oncogenic HRAS (T24) and other with a wild-type HRAS (RT4). The surviving fraction after exposure to 2 Gy of radiation (SF2) for the T24 cell lines was found to be 0.62, whereas that for RT4 cells was 0.40. Treatment with the farnesyl transferase inhibitor (FTI) L744,832, which inhibits RAS processing and activity, decreased the SF2 of T24 cells to 0.29, whereas the SF2 of RT4 cells remained unchanged after FTI treatment, thus demonstrating the importance of RAS activation to the radiosensitivity of cells with mutated RAS. MAP kinase activation was found to be constitutive and dependent on RAS in T24 cells, while it was inducible by radiation and was independent of RAS in RT4 cells. Treatment of both cell lines with the MAP2K inhibitor PD98059 inhibited MAPK activation; however, inhibiting MAPK activation had no effect on radiation survival of T24 or RT4 cells. These data indicate that MAPK activation does not contribute to RAS-induced radioresistance in this system

Radiation sensitization of human cancer cells in vivo by inhibiting the activity of PI3K using LY294002.

Multiple genetic alterations such as in Ras or EGFR can result in sustained signaling through PI3K. Our previous experiments have shown that resistance to radiation results from PI3K activity in cells in culture. Here we examined whether inhibition of PI3K in vivo would sensitize tumors to radiation. The human bladder cancer cell line T24 has amplified and mutated H-Ras resulting in sustained PI3K activity and phosphorylation of the downstream target of PI3K, Akt. Nude mice bearing T24 tumor cell xenografts were randomly assigned to one of four groups: control, radiation alone, the PI3K inhibitor LY294002 alone, or combined LY294002 and radiation. The LY294002 was delivered intraperitoneally to the mice. Downregulation of Akt was documented by Western blot analysis of tumor lysates. In vivo sensitization was measured using clonogenic assays or regrowth assays.A dose of 100 mg/kg of LY294002, but not 50 mg/kg, consistently eliminated the phosphorylation of Akt. This inhibition was transient, and Akt activity returned after 30 min. This dose resulted in severe respiratory depression and lethargy resolving without lethality. It is not possible to tell whether these side effects of LY294002 were mechanism-based or idiosyncratic. The PI3K inhibitor LY294002 by itself had minimal antitumor effect. The combination of LY294002 and radiation resulted in significant and synergistic reduction in clonogenicity and growth delay. Inhibition of PI3K by LY294002 can synergistically enhance radiation efficacy. This acts as a proof of principle that inhibition of the Ras to PI3K pathway could be useful clinically

The Ras radiation resistance pathway.

The critical pathways determining the resistance of tumor cells to ionizing radiation are poorly defined. Because the ras oncogene, a gene activated in many human cancers treated with radiotherapy, can induce increased radioresistance, we have asked which Ras effector pathways are significant in conferring a survival advantage to tumor cells. The phosphoinositide-3-kinase (PI3K) inhibitor LY294002 radiosensitized cells bearing mutant ras oncogenes, but the survival of cells with wild-type ras was not affected. Inhibition of the PI3K downstream target p70S6K by rapamycin, the Raf-MEK-MAPK pathway with PD98059, or the Ras-MEK kinase-p38 pathway with SB203580 had no effect on radiation survival in cells with oncogenic ras. Expression of active PI3K in cells with wild-type ras resulted in increased radiation resistance that could be inhibited by LY294002. These experiments have indicated the importance of PI3K in mediating enhanced radioresistance and have implicated PI3K as a potential target for specific radiosensitization of tumors

Direct evidence for the contribution of activated N-ras and K-ras oncogenes to increased intrinsic radiation resistance in human tumor cell lines.

Transformation with ras oncogenes results in increased radiation sur vival in many but not all cells. In addition, prenyltransferase inhibitors which inhibit ras proteins by blocking posttranslational modification radiosensitize cells with oncogenic ras. These findings suggest that oncogenic ras contributes to intrinsic radiation resistance. However, because introduction of ras oncogenes does not increase radiation survival in all cells and because prenyltransferase inhibitors target molecules other than ras, these studies left the conclusion that ras increases the intrinsic radi ation resistance of tumor cells in doubt. Here we show that genetic inactivation of K- or N-ras oncogenes in human tumor cells (DLD-1 and HT1080, respectively) leads to increased radiosensitivity. Reintroduction of the activated N-ras gene into the HT1080 line, having lost its mutant allele, resulted in increased radiation resistance. This study lends further support to the hypothesis that expression of activated ras can contribute to intrinsic radiation resistance in human tumor cells and extends this finding to the K- and N- members of the ras family. These findings support the development of strategies that target ras for inactivation in the treatment of cancer

Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction.

PURPOSE: Locoregional recurrence is the dominant form of treatment failure in head and neck (HandN) cancer. The epidermal growth factor receptor (EGFR) is frequently amplified in this disease