Vincent J. Bakanauskas

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 role of the H-ras oncogene in radiation resistance and metastasis

The sensitivity of tumor cells to the killing effects of ionizing radiation is thought to be one of the major determinants of curability of tumors in patients treated with radiation therapy. This paper reviews the evidence from our laboratory and other groups which supports a role for oncogenes in the induction of radioresistance in cultured mammalian cells. Primary rat embryo cells (REC) were chosen as a model system in which the effects on radiation resistance of the H-ras oncogene could be studied on a uniform genetic background. These cells offered several useful advantages. The cells prior to transformation are diploid and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any preexisting mutation affecting radiation response could be present. Additionally, the use of REC permitted the study of the effects of synergism between oncogenes on the induction of the radioresistant phenotype. The results show that the activated H-ras oncogene induces radiation resistance in primary rat cells after transformation, but that the effect of the oncogene itself is small. However, the myc oncogene, which has no effect on radiation resistance by itself, appears to have a synergistic effect on the induction of radiation resistance by H-ras. Radiation resistance induced by H-ras plus myc is characterized by an increase in the slope of the curve at high doses but there is also a large effect within the shoulder region of the radiation survival curve. The AdenoE1A oncogene which will also act synergistically with ras in transformation assays plays a less clear-cut role in assays of radiation resistance. The H-ras oncogene is also known not only to transform cells but also to induce metastatic behavior in the tumors which form after these transformed cells are injected into syngeneic animals or nude mice. We have also shown in our primary rat embryo cell system that the induction of metastatic behavior in transformed cells, like the induction of radioresistance depends on a complex interaction between oncogenes and the cellular background. This evidence will be reviewed to demonstrate some of the analogies between radiation resistance and metastasis as examples of the complex alterations in cellular phenotype which occur after oncogene transfection.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Abstract Gupta, A. K., Bernhard, E. J., Bakanauskas, V. J., Wu, J., Muschel, R. J. and McKenna, W. G. RAS-Mediated Radiation Resistance is not Linked to MAP Kinase Activation. 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.

Ras regulation of radioresistance in cell culture

Publisher Summary This chapter describes the Ras regulation of radioresistance in cell culture. Some tumor cells have intrinsic resistance to killing by ionizing radiation and this may limit the effectiveness of radiation in cancer treatment. As many as 20% of patients who present with localized disease fail because of uncontrolled disease at the primary site, without signs of disseminated disease. One factor known to increase tumor cell resistance to radiation is the presence of activated oncogenes. Transfection with ras oncogenes is shown to increase radioresistance in certain rodent and human cells, although increased radioresistance was not seen in all cell types after ras transfection. Ras proteins are processed in a series of reactions that result in farnesylation or geranylgeranylation by farnesyltransferase or geranylgeranyltransferase, respectively. This is essential for the attachment of Ras to the inner surface of the plasma membrane and for activity. There is now ample evidence that Ras mutations contribute to radiation resistance in human cell lines. Activating mutations of ras can be seen in 30% of all human tumors.

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 mycV. 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. Key Words: c-Raf-1, Ras, Transformation

32 C-RAF-1 protein kinase is not essential for ras transformation in 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.

Features of Tumor Progression in H- ras transformed Rat Embryo Cells

The initial description of oncogenes was based on the ability of modified cellular genes to induce transformation in tissue culture or tumorigenicity when cells expressing those genes were introduced into animals. Since the ability of a tumor to metastasize is an additional property of a tumor cell, distinct from the formation of a tumor (Fidler and Hart, 1982; Kripke et al., 1978), one might expect that oncogenes would play no role in metastasis or in tumor progression in general. On the other hand, activated ras oncogenes are found frequently in some types of human carcinoma such as colon (Vogelstein and Gillespie, 1979) and pancreatic carcinoma (Almoguera et al., 1988) and in the case of colorectal carcinoma the activation of ras may be a late event in the development of the tumor, suggesting that the actual effect of ras may be to influence tumor progression rather than the initiation event. Thus, studies of the effect of oncogenes upon metastasis and other parameters of tumor progression as studied in the laboratory might help to evaluate the potential influence these genes may have in tumor progression in vivo.