Transferrin receptor targeting nanomedicine delivering wild-type p53 gene sensitizes pancreatic cancer to gemcitabine therapy.

To overcome gene therapy barriers such as low transfection efficiency and nonspecific delivery, liposomal nanoparticles targeted by a single-chain antibody fragment to the transferrin receptor (TfRscFv) delivering wild-type (wt) human p53 (SGT-53) were developed for tumor-specific targeting. We hypothesize that SGT-53 in combination with gemcitabine will demonstrate enhanced therapeutic benefit in an in vivo metastatic pancreatic cancer model. Intrasplenic injection of 1 × 10(6) Panc02 murine pancreatic cancer cells was used to generate in vivo hepatic metastatic tumors. Nanoparticle localization was assessed by tail vein injection of TfRscFv with fluorescently labeled oligonucleotides (6-carboxyfluorescein phosphoramidite (6FAM) ODN) imaged by Xenogen IVIS 200 scan. SGT-53 (equivalent to 30 µg of p53 intravenously) and gemcitabine (20 mg/kg intraperitoneally) alone and in combination were administered biweekly and compared with untreated mice. Survival was determined by blinded daily assessment of morbidity. Human wtp53 expression and transferrin levels in the tumors were assessed by western blot analysis. Tumor burden was quantified by liver weight. Xenogen imaging demonstrated tumor-specific uptake of TfRscFv-6FAM ODN. Exogenous human wtp53 protein was detected in the SGT-53-treated tumors compared with control. Compared with untreated mice with metastatic tumors demonstrating median survival of 20 days, SGT-53, gemcitabine and the combination demonstrated improved median survival of 29, 30 and 37 days, respectively. The combination treatment prolonged median survival when compared with single drug treatment and decreased tumor burden. The tumor targeting liposomal-based SGT-53 nanoparticle is capable of sensitizing pancreatic cancer to conventional chemotherapy in pancreatic cancer models. This approach has the potential to be translated into a new, more effective therapy for pancreatic cancer. Further optimization is ongoing, moving towards a Phase 1B/2 clinical trial.

T-cell vaccines that elicit effective immune responses against HCV in chimpanzees may create greater immune pressure for viral mutation.

A prime/boost vaccine strategy that transfects antigen-presenting cells using ligand-modified immunoliposomes to efficiently deliver plasmid DNA, followed by boosting with non-replicating recombinant adenovirus was used in chimpanzees to generate HCV-specific memory T-cells. Three chimpanzees (two vaccines, one control) were immunized with immunoliposomes complexed with DNA expressing NS3-NS5B or complexed with empty vector. Animals were boosted with adenovirus expressing NS3-NS5B, or non-recombinant adenovirus (control). Using liposome delivery we were able to obtain specific HCV responses following DNA priming in the chimpanzees. This data and mouse immunization studies confirm this as a more efficient delivery system than direct intramuscular inoculations with naked DNA. Subsequent to the adenovirus boost significant increases in peripheral HCV-specific T-cell responses and intrahepatic IFN-gamma and CD3varepsilon mRNA were also observed in the two vaccinated animals. Following challenge (100 CID(50)) both vaccinated animals showed immediate and significant control of viral replication (peak titers 3.7x10(4) and 9x10(3)IU/mL at weeks 1 and 2), which coincided with increases in HCV-specific T-cell responses. Viral kinetics in the control animal were comparable to historical controls with exponential increases in titer during the first several weeks. One vaccinated animal developed a low-level persistent infection (2x10(3)IU/mL) which correlated with a decrease in HCV-specific T-cell responses. Circulating virus isolated from both vaccinated animals showed approximately 2-fold greater nonsynonymous mutation rates compared to controls and the nonsynonymous/synonymous mutation rate ratio was indicative of positive selection. These data suggest that although T-cell vaccines can induce immune responses capable of controlling HCV, they also induce high levels of immune pressure for the potential selection of escape mutants.

Physical characterization methods for iron oxide contrast agents encapsulated within a targeted liposome-based delivery system.

Intact liposome-based targeted nanoparticle delivery systems (NDS) are immobilized by non-selective binding and characterized by scanning probe microscopy (SPM) in a fluid imaging environment. The size, size distribution, functionality, and stability of an NDS with a payload consisting of a super-paramagnetic iron oxide contrast agent for magnetic resonance imaging are determined. SPM results are combined with information obtained by more familiar techniques such as superconducting quantum interference device (SQUID) magnetometry, dynamic light scattering, and electron microscopy. By integrating the methods presented in this work into the NDS formulation and manufacturing process, size-dependent statistical properties of the complex can be obtained and the structure-function relationship of individual, multi-component nanoscale entities can be assessed in a reliable and reproducible manner.

A sterically stabilized immunolipoplex for systemic administration of a therapeutic gene.

A sterically stabilized immunolipoplex (TsPLP), containing an antitransferrin receptor single-chain antibody fragment (TfRscFv)-PEG molecule, has been developed to specifically and efficiently deliver a therapeutic gene to tumor cells. A postcoating preparation strategy was employed in which a DNA/lipid complex (lipoplex) was formed first and then sequentially conjugated with PEG and TfRscFv. The complex prepared by this method was shown to be superior in ability to deliver genes to tumor cells than when prepared by a common precoating strategy, in which DNA is mixed with TfRscFv-PEG conjugated liposome. Using prostate cancer cell line DU145, a comparison was made between the in vitro and in vivo gene delivery efficiencies of four complexes, Lipoplex (LP), PEG-Lipoplex (PLP), TfRscFv-PEG-Lipoplex (TsPLP) and our standard TfRscFv-Lipoplex (TsLP). In vitro, the order of transfection efficiency was TsLP>LP approximately TsPLP>PLP. However, in vivo the order of transfection efficiency, after systemic administration via the tail vein, was TsPLP>TsLP>LP or PLP with TsPLP-mediated exogenous gene expression in tumor being two-fold higher than when mediated by TsLP. This suggests that the in vitro transfection efficiency of TsPLP was not indicative of its in vivo efficiency. In addition, it was found that the level of exogenous gene expression in the tumor mediated by TsPLP was higher than that mediated by TsLP and did not decrease over the time. More importantly, high exogenous gene expression in tumor, but low expression in liver, was observed after an i.v. delivery of TsPLP carrying either the GFP reporter gene or the p53 gene, indicating that tumor preferential targeting was maintained by this complex in the presence of PEG. These findings show that incorporation of PEG into our targeted lipoplex results in a more efficient delivery of the complex to the tumor cells, possibly by inhibiting the first pass clearance observed with non-PEG containing liposomes. Therefore, these data demonstrate that TsPLP is a improvement over our previously established tumor targeted gene delivery complex for systemic gene therapy of cancer.

Inhibitory effects of the combination of HER-2 antisense oligonucleotide and chemotherapeutic agents used for the treatment of human breast cancer.

Poor response to chemotherapy in patients with breast cancer is often associated with overexpression of HER-2/neu. Interference with HER-2 mRNA translation by means of antisense oligonucleotides might improve the efficacy of chemotherapy. To test this hypothesis, eight breast cancer cell lines and a normal human fibroblast cell line were examined for their level of HER-2 expression, their sensitivity to phosphorothioate antisense oligonucleotides (AS HER-2 ODN), and to various chemotherapeutic agents, and the combination of the two. No correlation was found between the intrinsic HER-2 level and either the sensitivity to a particular chemotherapeutic agent alone, or the amount of growth inhibition observed with a specific AS HER-2 ODN concentration. Although sequence specificity and extent of AS HER-2 ODN inhibition of HER-2 synthesis were somewhat higher in the HER-2 overexpressing MDA-MB-453 and SK-BR-3 cells, we found that antisense treatment significantly sensitized all of the breast cancer cells, even MDA-MB-231 and MDA-MB-435 cells, with approximately basal levels of HER-2, to various chemotherapeutic agents. In addition, the combination of AS HER-2 ODN and taxol was shown to synergistically induce apoptosis in MDA-MB-435. These results demonstrate that overexpression of HER-2 would not be a prerequisite for the effective use of AS HER-2 ODN as a combination treatment modality for breast cancer and suggest that the use of AS HER-2 ODN, as part of a combination treatment modality, need not be limited to breast tumors that display elevated levels of HER-2.

Downmodulation of bFGF-binding protein expression following restoration of p53 function.

Angiogenesis is a requirement for solid tumor growth. Therefore, inhibition of this neovascularization is one mechanism by which restoration of wtp53 function may lead to tumor regression. Here we report that adenoviral vector-mediated wild-type p53 transduction results in growth inhibition of squamous cell carcinoma of the head and neck tumor cells both in vitro and in a xenograft mouse model. This growth inhibition is associated with the down-regulation of the expression of fibroblast growth factor binding protein, a secreted protein required for the activation of angiogenic factor basic FGF. These findings suggest that wtp53-induced tumor regression is due, at least in part, to antiangiogenesis mediated by the downmodulation of fibroblast growth factor binding protein.

Systemic p53 gene therapy of cancer with immunolipoplexes targeted by anti-transferrin receptor scFv.

BACKGROUND: A long-standing goal in genetic therapy for cancer is a systemic gene delivery system that selectively targets tumor cells, including metastases. Here we describe a novel cationic immunolipoplex system that shows high in vivo gene transfer efficiency and anti- tumor efficacy when used for systemic p53 gene therapy of cancer. MATERIALS AND METHODS: A cationic immunolipoplex incorporating a biosynthetically lipid-tagged, anti-transferrin receptor single-chain antibody (TfRscFv), was designed to target tumor cells both in vitro and in vivo. A human breast cancer metastasis model was employed to evaluate the in vivo efficacy of systemically administered, TfRscFv-immunolipoplex-mediated, p53 gene therapy in combination with docetaxel. RESULTS: The TfRscFv-targeting cationic immunolipoplex had a size of 60-100 nm, showed enhanced tumor cell binding, and improved targeted gene delivery and transfection efficiencies, both in vitro and in vivo. The p53 tumor suppressor gene was not only systemically delivered by the immunolipoplex to human tumor xenografts in nude mice but also functionally expressed. In the nude mouse breast cancer metastasis model, the combination of the p53 gene delivered by the systemic administration of the TfRscFv-immunolipoplex and docetaxel resulted in significantly improved efficacy with prolonged survival. CONCLUSIONS: This is the first report using scFv-targeting immunolipoplexes for systemic gene therapy. The TfRscFv has a number of advantages over the transferrin (Tf) molecule itself: (1) scFv has a much smaller size than Tf producing a smaller immunolipoplex giving better penetration into solid tumors; (2) unlike Tf, the scFv is a recombinant protein, not a blood product; (3) large scale production and strict quality control of the recombinant scFv, as well as scFv-immunolipoplex, are feasible. The sensitization of tumors to chemotherapy by this tumor-targeted and efficient p53 gene delivery method could lower the effective dose of the drug, correspondingly lessening the severe side effects, while decreasing the possibility of recurrence. Moreover, this approach is applicable to both primary and recurrent tumors, and more significantly, metastatic disease. The TfRscFv-targeting of cationic immunolipoplexes is a promising method of tumor targeted gene delivery that can be used for systemic gene therapy of cancer with the potential to critically impact the clinical management of cancer.

Tumor-targeted p53-gene therapy enhances the efficacy of conventional chemo/radiotherapy.

A long-standing goal in gene therapy for cancer is a stable, low toxic, systemic gene delivery system that selectively targets tumor cells, including metastatic disease. Progress has been made toward developing non-viral, pharmaceutical formulations of genes for in vivo human therapy, particularly cationic liposome-mediated gene transfer systems. Ligand-directed tumor targeting of cationic liposome-DNA complexes (lipoplexes) is showing promise for targeted gene delivery and systemic gene therapy. Lipoplexes directed by ligands such as folate, transferrin or anti-transferrin receptor scFv, showed tumor-targeted gene delivery and expression in human breast, prostate, head and neck cancers. The two elements, ligand/receptor and liposome composition, work together to realize the goal of functional tumor targeting of gene therapeutics. The tumor suppressor gene, p53, has been shown to be involved in the control of DNA damage-induced apoptosis. Loss or malfunction of this p53-mediated apoptotic pathway has been proposed as one mechanism by which tumors become resistant to chemotherapy or radiation. The systemically delivered ligand-liposome-p53 gene therapeutics resulted in efficient expression of functional wild-type p53, sensitizing the tumors to chemotherapy and radiotherapy. This is a novel strategy combining current molecular medicine with conventional chemotherapy and radiotherapy for the treatment of cancer. The systemic delivery of normal tumor suppressor gene p53 by a non-viral, tumor-targeted delivery system as a new therapeutic intervention has the potential to critically impact the clinical management of cancer.

Does p53 status influence tumor response to anticancer therapies?

Abnormalities in the tumor suppressor gene p53 have been identified in over 60% of human cancers. Since it plays such a pivotal role in cell growth regulation and apoptosis, the status of the p53 gene has been proposed as one of the major determinants of a tumor's response to anticancer therapies. In this review we examine the relationship between functional p53 and sensitivity/resistance to both chemotherapy and radiotherapy, and discuss the potential use of some of the current gene therapy approaches to restore functional p53 to tumors as a means of modulating the effects of radiation and chemotherapy.

Tp53 gene therapy: a key to modulating resistance to anticancer therapies?

Abnormalities in the p53 tumor suppressor have been identified in over 60% of human cancers. The status of p53 within tumor cells has been proposed to be one of the major determinants of the response to anticancer therapies. In this review we examine the relationship between functional p53 and sensitivity, or resistance, to chemotherapy and radiotherapy. We also discuss the potential of current gene-therapy approaches to restore functional p53 to tumors as a means of modulating the effects of radiation and chemotherapy.

3'-End conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol: synthesis and anti-ras activity in radiation-resistant cells.

Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with all-phosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3'-end (PPS-C(16)). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3'- and 5'-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS-C(16) ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS-C(16) ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS-C(16) ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.

Non-viral gene delivery for p53.

Abnormality in the tumor suppressor gene p53 is one of the most common occurrences associated with human neoplasia. Consequently, restoration of wild-type p53 function is seen as a particularly promising approach for cancer gene therapy. In recent years, considerable research effort has centered upon developing and improving non-viral delivery systems as alternatives to viral vectors for gene delivery. These methods include the use of lipoplexes and polyplexes, and even delivery of naked DNA. Optimally effective cancer gene therapy requires treatment of metastatic as well as local disease, and to achieve this end, systemic delivery systems for therapeutic genes will be required. This review will discuss some of the recent advances in ways to improve targeting, transfection efficiency and stability for systemic, non-viral p53 gene therapy.

Transferrin-liposome-mediated systemic p53 gene therapy in combination with radiation results in regression of human head and neck cancer xenografts.

The use of cationic liposomes as nonviral vehicles for the delivery of therapeutic molecules is becoming increasingly prevalent in the field of gene therapy. We have previously demonstrated that the use of the transferrin ligand (Tf) to target a cationic liposome delivery system resulted in a significant increase in the transfection efficiency of the complex [Xu, L., Pirollo, K.F., and Chang, E.H. (1997). Hum. Gene Ther. 8, 467-475]. Delivery of wild-type (wt) p53 to a radiation-resistant squamous cell carcinoma of the head and neck (SCCHN) cell line via this ligand-targeted, liposome complex was also able to revert the radiation resistant phenotype of these cells in vitro. Here we optimized the Tf/liposome/DNA ratio of the complex (LipT) for maximum tumor cell targeting, even in the presence of serum. The efficient reestablishment of wtp53 function in these SCCHN tumor cells in vitro, via the LipT complex, restored the apoptotic pathway, resulting in a significant increase in radiation-induced apoptosis that was directly proportional to the level of exogenous wtp53 in the tumor cells. More significantly, intravenous administration of LipT-p53 markedly sensitized established SCCHN nude mouse xenograft tumors to radiotherapy. The combination of systemic LipT-p53 gene therapy and radiation resulted in complete tumor regression and inhibition of their recurrence even 6 months after the end of all treatment. These results indicate that this tumor-specific, ligand-liposome delivery system for p53 gene therapy, when used in concert with conventional radiotherapy, can provide a new and more effective means of cancer treatment.

Restoration of the G1 checkpoint and the apoptotic pathway mediated by wild-type p53 sensitizes squamous cell carcinoma of the head and neck to radiotherapy.

BACKGROUND: A significant number of squamous cell carcinomas of the head and neck (SCCHN) resist radiation treatment, the most common form of adjuvant therapy for this disease. The presence of a mutant form of the tumor suppressor gene p53 has been correlated with disruption of programmed cell death (apoptosis) and reduced cell cycle arrest, resulting in increased radiation resistance and survival. METHODS AND RESULTS: We introduced by means of an adenoviral vector a functional p53 gene into a radiation-resistant SCCHN cell line that harbors mutant p53. Replacement of wild-type p53 restored the G1 block and apoptosis in these cells in vitro. Moreover, introduction of wild-type p53 sensitized SCCHN-induced mouse xenografts to radiotherapy in vivo. CONCLUSION: The combination of p53 replacement gene therapy with conventional radiotherapy may treat SCCHN more effectively.

p53 mediated sensitization of squamous cell carcinoma of the head and neck to radiotherapy.

Radiation resistant squamous cell carcinoma of the head and neck cell line JSQ-3 carries a mutant form of tumor suppressor gene p53. Treatment of these cells with an adenoviral vector containing wild-type p53 (Av1p53) was able to inhibit their growth in vitro and in vivo while having no effect on normal cells. More significantly, introduction of wtp53 also reduced the radiation-resistance level of this cell line in vitro, in a viral dose-dependent manner. Furthermore, this radiosensitization also carried over to the in vivo situation where the response of JSQ-3 cell-induced mouse xenografts to radiotherapy was markedly enhanced after treatment with Av1p53. Complete, long-term regression of the tumors for up to 162 days was observed when a single dose of Av1p53 was administered in combination with ionizing radiation, demonstrating the effectiveness of this combination of gene therapy and conventional radiotherapy. This sensitization of tumors to radiation therapy by replacement of wtp53 could significantly decrease the rate of recurrence after radiation treatment. Since radiation is one of the most prevalent forms of adjunctive therapy for a variety of cancers, these results have great relevance in moving toward an improved cancer therapy.

Transferrin-liposome-mediated p53 sensitization of squamous cell carcinoma of the head and neck to radiation in vitro.

Wild-type (wt) p53 DNA was transfected into the radioresistant human cell line JSQ-3, established from a squamous cell carcinoma of the head and neck (SCCHN), using a transferrin-liposome system, and the ability of the introduced wt p53 to sensitize the transfected JSQ-3 cells to ionizing radiation was examined. Transferrin increased the in vitro transfection efficiency of cationic liposomes up to 70-80% in JSQ-3 cells, representing a 6- to 10-fold increase over liposome transfection alone. The exogenous wt p53 was expressed at high levels in transferrin-liposome-DNA-transfected cells and resulted in the reversion of the radioresistant phenotype of the JSQ-3 cells in a DNA dose-dependent manner. The D10 values were reduced from 6.36 +/- 0.54 Gy to 4.13 +/- 0.06 Gy, a value in the radiosensitive range. In vivo, the intratumoral injection of the transferrin-liposome system resulted in a higher number of transfected tumor cells in the JSQ-3 induced nude mouse xenografts when compared with transfection by liposome alone. The results indicate that the combination of p53 replacement gene transduction, mediated by the relatively safe transferrin-liposome system, and conventional ionizing radiation may provide a more effective treatment for head and neck cancer.

Evidence supporting a signal transduction pathway leading to the radiation-resistant phenotype in human tumor cells.

A signal transduction pathway, involving oncogenes and their normal counterparts the proto-oncogenes, analogous to that for cell growth and differentiation has been proposed to lead to the phenotype of cellular radioresistance (RR). In this report we provide evidence demonstrating the existence of such a pathway by using antisense oligonucleotides (ASO) to reverse the RR phenotype. Utilizing ASO directed against the raf-1 gene, a central component of this proposed pathway, we were able to reverse the RR phenotype of human tumor cell lines having elevated HER-2 expression or a mutant form of Ha-ras, two genes upstream of raf-1 in signal transduction. Additionally, anti-ras ASO were able to radiosensitize HER-2 overexpressing cells. These results, which verify the presence of a signaling pathway leading to cellular RR, also have possible clinical implications for the use of ASO as a means to sensitize radioresistant tumors to radiation therapy.

Cytogenetic response to G2-phase X irradiation in relation to DNA repair and radiosensitivity in a cancer-prone family with Li-Fraumeni syndrome.

Noncancerous skin fibroblasts from six family members with Li-Fraumeni syndrome, five with cancer of diverse tissue origin and one with a premalignant neoplasm, showed a high frequency of chromatid aberrations, 94 to 119 breaks and 58 to 95 gaps per 100 metaphase cells arrested with colcemid 0.5 to 1.5 h after X irradiation (1.75 x 10(-2) C/kg). This response results from deficient repair of the radiation-induced DNA damage. In contrast, skin fibroblasts from two unrelated normal controls and a spouse showed 19 breaks and 17 to 19 gaps per 100 cells. Whereas all six members of the cancer-prone family had a radioresistant phenotype, only four had an inherited p53 mutation. Fibroblasts from a radioresistant family member showed the same extent of chromatid damage directly (0 to 0.5 h) after G2-phase X irradiation as those from the radiosensitive control spouse. We conclude, therefore, that radiosensitivity, as determined by cell killing in asynchronous populations of skin fibroblasts, is unrelated to chromosomal sensitivity to G2-phase X irradiation. However, the persistence of a high frequency of chromatid breaks and gaps at 0.5 to 1.5 h after G2-phase X irradiation, a manifestation of deficient DNA repair, is associated with proneness to cancer in this family.

Several mutant p53 proteins detected in cancer-prone families with Li-Fraumeni syndrome exhibit transdominant effects on the biochemical properties of the wild-type p53.

The identification of germ-line mutations in the p53 gene has provided a situation where comparable amounts of wild-type and mutant p53 co-exist in constitutional cells of certain individuals who are cancer-prone. Here we report the biochemical characteristics of several Li-Fraumeni syndrome associated mutant p53 proteins in order to assess the influence of germ-line mutant p53 on the functions of the wild-type p53. Unlike 248W mutant p53 protein, which was previously shown to have no effect on the wild-type p53 conformation (Milner & Medcalf, 1991; Cell 65, 765-774), germ-line associated mutant p53 proteins with residue 133T, 245D or 258K, converted the wild-type p53 conformation into the mutant conformation. Furthermore, lysates containing cotranslated wild-type p53 and these mutant p53 proteins were significantly impaired for DNA and SV40 large T antigen binding. These observations suggest that at least some germ-line p53 mutants might exhibit dominant effects on wild-type p53 functions and, like other mutant p53 proteins, the phenotype of germ-line mt p53 proteins might be variable depending on the particular mutation.

Oncogene- transformed NIH 3T3 cells display radiation resistance levels indicative of a signal transduction pathway leading to the radiation-resistant phenotype.

Oncogenes and their normal counterparts, proto-oncogenes, are functionally important cellular genes which interact with one another as components of signal transduction pathways leading to cell growth and differentiation. Numerous reports in the literature have also begun to link these genes to the phenomenon of cellular radiation resistance. In this report we examine the radiation resistance level of NIH 3T3 cells transformed by various oncogenes in an attempt to define the intracellular pathway to the radiation-resistant phenotype. The results demonstrate that an analogous signaling pathway is apparently involved in acquisition of radiation resistance. Serine/threonine protein kinase oncogenes such as raf, mos, and PKC play a central role in the pathway. Moreover, specific oncogenes upstream (sis, HER-2, met, trk, and ras) and downstream (ets and myc) of these important signaling mediators can also influence the radiation resistance level of the cells.