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Survivin increases resistance to radiation-induced cell death and is upregulated by AKT signaling in primary human glioblastoma cell lines
174
I. J. Radiation Oncology
1027
● Biology ● Physics
Volume 54, Number 2, Supplement, 2002
Transducing Tat-Anti-MDM2 Peptides Enhances Cytotoxic Effect of Radiation and Chemotherapy on Hepatoma Cells IN VITRO
J. Cho, Y. Du, H. Dai, K. Chao Department of Radiation Oncology, Washington University, Saint Louis, MO Purpose/Objective: MDM-2, a negative regulator p53 tumor suppressor protein, is a potential target for developing novel molecular-targeted single and combined modality therapy for tumors expressing MDM2 and wild type p53. Hepatocellular carcinoma is known to be resistant to existing therapeutic approaches and therefore the prognosis remains poor. Here we examined the efficacy of a synthetic transducing Tat-␣MDM2 peptide and its combination with radiation and/or chemotherapy on the killing of hepatoma cells. Materials/Methods: We first assessed the biodistribution of Tat-peptide in vitro and in mouse organs. Cellular uptake and localization of Tat conjugated peptides were assessed using FITC labeled peptides after intra-peritoneal and intra-venous injection. Mouse hepatoma cell line (Hepa1c1c7) and mouse normal hepatocytes (BNL-CL2), both expressing wild-type p53, were used. The 12-aa MDM-2 binding domain of mouse p53 protein (QETFSGLWKLLP) was fused with a 12-aa transducing domain of HIV Tat protein at N-terminal during synthesis to create Tat-␣MDM2. Tat-peptide alone or a mutant form of the Tat-␣MDM2 peptide, which has the three MDM-2 interacting residues changed to Alanine, was served as the controls. Cell viability was quantified by clonogenic assay, colorimetric microplate MTS assay, or trypan blue exclusion test. Apototic cell death was confirmed by a colorimetric caspase-3 assay with CaspACETM using Ac-DEVE-p-nitroanilide as substrate and DNA breakage by TUNEL assay using fluorseine ApopTag威. Results: In cultured mouse and rat cells, cellular fluorescence peaks within 25 min after the addition of FITC peptides, with significant accumulation in both nuclei and cytoplasma. The distribution of FITC-Tat was assessed in vivo in nude mice bearing tumor xenografts on either flank. One hour after intra-peritoneal and intra-venous injection, substantial amount of FITC was detected in the subcutaneous tumors. Distribution to mouse liver is the most significant one among organs examined. This implicates the potential capability of this transducing Tat-Peptide in reaching tumor cells residing within the liver. We then turned to examine tumor-killing efficacy of Tat-␣MDM2 on hepatoma cells. The IC50 and IC90 for Tat-␣MDM2 were 300 mM and 500 mM, respectively for Hepa1c1c7. Tat alone or Tat-␣MDM2-mutant did not induce Hepa1c1c7 cell death at concentration up to 500 mM. No toxicity was observed for BNL-C2 cells with all peptides tested at up to 500 mM. Pretreatment with 150 mM Tat-␣MDM2, which did not produce detectable cell death when given alone, reduced the percent survival from 80% to 37% for Hepa1c1c7 cells irradiated at single dose of 5Gy. Similarly, pretreating Hepa1c1c7 with 150 mM Tat-␣MDM2 also lowered the IC50 for cisplatin from 25 mM to 11 mM. A 3.3- to 4.8-fold induction of p53 reporter luciferase activity was detected by Tat-␣MDM2 treatment in Hepa1c1c7 cells. Hepatoma cells treated with Tat-␣MDM2 stained strongly positive in TUNEL assay; however, Z-VAD-fmk did not protect Hepa1c1c7 cells against the killing effect of Tat-␣MDM2. Further, no caspase-3 activity was detected in cells treated with 500 mM Tat-␣MDM2, in contrast to 17 pmol pNA/mg/hour in those treated with 2 mM camptothecin. One possible factor that may have obscured the investigation of down-stream cell death paradigm is the rapid death of Hepa1c1c7. By trypan blue dye exclusion assay, percent cell death was 50%, 80%, and 99% at 30 min, 1 hour, and 2 hour, respectively, after 500 mM Tat-␣MDM2 treatment. Conclusions: Transducing Tat-peptide is capable of reaching cells in the liver after intra-peritoneal and intra-venous injection. We have shown that Tat-␣MDM2 peptide was selectively cytotoxic to hepatoma cells, and sublethal dose of Tat-␣MDM2 enhanced the cytotoxic effect of ionizing radiation and chemotherapy agent (cisplatin). With these encouraging in vitro results, we are proceeding with In Vivo investigation.
1028
Survivin Increases Resistance to Radiation-Induced Cell Death and is Upregulated by AKT Signaling in Primary Human Glioblastoma Cell Lines
A. Chakravarti, A. Chaklader, D.E. Latham, M.A. Delaney, J.S. Loeffler Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, Boston, MA Purpose/Objective: Survivin is a novel antiapoptotic gene that has been recently cloned and characterized. Its expression has been found to be of prognostic value in several tumor types, including human gliomas. Although Survivin has been found to suppress apoptotic cell death, its ability to inhibit radiation-induced cell death has been less well characterized. The purpose of this study was to investigate whether Survivin has a role in inhibiting radiation-induced cell death and, if so, to further investigate the underlying mechanism(s) involved. Materials/Methods: Two primary, tumor-derived, glioblastoma (GBM) cell lines were established in vitro and were both found to express Survivin by Western blot and by RT-PCR analysis. Inactivation of Survivin was accomplished in multiple ways: using antisense oligonucleotides, transient transfection experiments using dominant-negative constructs (nonphorylatable Thr(34)- Ala mutant called T34A), and by adenoviral vectors containing dominant-negative Survivin (Adeno-T34A). Subsequently, the cells were irradiated in doses ranging from 2 to 10 Gy, and survival was assessed using time-course apoptosis (Annexin V and cleaved caspase 3), viability, and clonogenic survival assays. As it was previously reported that AKT can upregulate Survivin levels in certain cell types, the AKT-Survivin relationship was also examined. Results: Inactivation of Survivin by any of these three methods significantly enhanced peak apoptosis values following radiation (P⬍0.0001) and reduced tumor cell viability and clonogencity (p⬍0.0001). Using selective pharmacologic inhibitors of caspase 3, it was determined that the reduction of the latter was, indeed, due to suppression of apoptosis. Interestingly, pharmacologic inhibition of caspase 8 reversed the enhanced cell death observed upon inactivation of Survivin, suggesting that Survivin can suppress both mitochondria-dependent and -independent apoptotic pathways. As would be expected, cultures of normal astrocytes failed to express Survivin, and Survivin inactivation failed to produce any demonstrable effects. Transient transfection experiments of GBM cell lines with dominant-negative, or kinase-deficient, AKT (AKT-KD), and with the constitutively active, or myristylated, form of AKT (AKT-Myr) suggest that AKT activity may be important in mediating accumulation of Survivin in the G1/S, as well as its known accumulation in the G2/M phases of the cell cycle.
Proceedings of the 44th Annual ASTRO Meeting
Conclusions: Survivin appears to enhance survival of GBM cells following radiation by suppressing both mitochondriadependent and -independent apoptotic pathways. Activation of AKT signaling appears to lead to accumulation of Survivin in the G1/S phases of the cell cycle, where it is normally absent. Given its exclusive expression in tumor cells, molecular targeting of Survivin appears to be a promising strategy to enhance the therapeutic gain of radiation in gliomas.
1029
Methylation of the Novel Apoptotic Gene TMS1/ASC in Human Glioblastoma Multiforme
1
W. Bobo , A. Stone1, D. Brat2, S.N. Devi3, E. Van Meir3, P.M. Vertino1 1 Department of Radiation Oncology, Emory University, Atlanta, GA, 2Department of Pathology, Emory University, Atlanta, GA, 3Department of Neurosurgery, Emory University, Atlanta, GA Purpose/Objective: Glioblastoma multiforme (GBM) is the most malignant of human brain tumors. Patients exhibit a mean survival of less than 12 months despite continued progress in neurosurgical techniques and the use adjuvant chemo- and radiation therapy. Defining the molecular events that contribute to the inititation and progression of these tumors, and an understanding of how these molecular events affect response to therapy are of critical importance in the tailoring of patient-specific treatment regimens. Acquired genetic and epigenetic alterations that lead to defects in apoptosis are common in human cancers, and promote tumorigenesis by allowing cells to survive under conditions that would normally trigger a cell death response. Unlike normal cells, cancer cells survive despite excessive DNA damage, in the absence of otherwise required growth factors, and under the hypoxic conditions that arise when a tumor outgrows its blood supply. Importantly, the propensity of tumor cells to undergo apoptosis is a critical determinant of their sensitivity to most chemotherapeutic agents and radiation. Therefore, defects in apoptotic signaling contribute to tumor initiation and progression, and can lead to drug resistance and treatment failure. Recently, we identified a novel gene, TMS1, that is silenced in association with aberrant DNA methylation in human breast and other cancers. TMS1 functions in the promotion of apoptosis, and acts upstream of caspase-9, a critical mediator of the apoptotic response to anticancer agents and other forms of cellular stress. Therefore, loss of TMS1 via aberrant methylation may contribute to human tumor development by allowing cells to bypass apoptosis and, as a consequence, may cause tumor cells to be resistant to anticancer drugs and radiation. The goals of the present study were to determine whether the TMS1 gene was aberrantly methylated in human glioblastomas, and to determine the relationship between TMS1 methylation and clinical disease course. Materials/Methods: Twenty-six human glioblastoma cell lines and twenty-five primary glioblastomas were evaluated for methylation of the TMS1 promoter using methylation-specific PCR. Tumor specimens were frozen immediately following surgical resection. All specimens were reviewed by a single neuropathologist for histologic confirmation of GBM and tissue integrity. Normal brain biopsies were obtained at autopsy from five cancer-free individuals. Expression of TMS1 was determined by reverse-transcriptase PCR. Results: We found that whereas normal brain tissue (5/5) was completely unmethylated at the TMS1 gene and expressed TMS1 mRNA, 10 of 25 (40%) of primary glioblastomas exhibited aberrant hypermethylation of the gene. In one case, there was complete methylation of the TMS1 gene. There was no correlation between methylation of TMS1 and patient age or sex. Interestingly, methylation of TMS1 tended to correlate with shorter post-operative survival times. Patients with tumors exhibiting an unmethylated TMS1 gene had a mean survival of 22 months (median⫽14) whereas those exhibiting aberrant methylation of the TMS1 gene had a mean survival of 9.6 months (median ⫽ 8). Conclusions: We are currently examining whether methylation of TMS1 may identify a subset of patients with reduced response to adjuvant chemo- or radiation therapy. Such findings would be consistent with the hypothesis that methylation of TMS1 confers resistance to cell death induced by chemotherapeutic agents and radiation, and would support the use of TMS1 methylation prognostic indicator for the treatment of glioblastoma.
1030
EGFR-CD533 Acts as a Pan-ERBB Receptor Tyrosine Kinase Inhibitor
J.N. Contessa, A. Abell, G. Lammering, T. Hewit, K. Valerie, R.K. Schmidt-Ullrich Department of Radiation Oncology, The Medical College of Virginia / Virginia Commonwealth University, Richmond, VA Purpose/Objective: The ERBB receptor tyrosine kinases (RTK), ERBB1 (EGFR), ERBB2, -3 and -4 are expressed at varied levels in human carcinoma and malignant glioma cells. These wild-type receptors as well as EGFRvIII, a constitutively active EGFR mutant lacking the extracellular N-terminal EGF binding domain, are involved in autocrine growth regulation of neoplastic cells. We have demonstrated that radiation induces indiscriminate activation of all ERBB species expressed by a given tumor cell which leads to powerful signals along the cytoprotective, mitogen-activated protein kinase (MAPK) pathway and confers radioresistance. Because of the nature of radiation-induced ERBB RTK activation only a pan-ERBB inhibitor can be expected to produce maximum radiosensitization. We have therefore investigated the effects of a dominant negative EGFR in blocking ERBB RTK signaling. Materials/Methods: The pan-ERBB inhibitory function of dominant negative EGFR-CD533, which lacks 533 C-terminal amino acids including the tyrosine kinase domain, is based upon the truncated receptor’s ability to interfere with ERBB RTK dimerizations and transactivation. We tested the effectiveness of the EGFR-CD533 in disrupting both growth factor and radiation-induced cytoprotective signals and its ability to confer radiosensitization using carcinoma and malignant glioma cell lines. Because of the ability to transduce human carcinomas with high efficiency and minimum toxicity, we employed an adenoviral vector, Ad-EGFR-CD533. The inhibitory effectiveness of EGFR-CD533 was studied in MDA-MB-231 and T47D breast carcinoma cell lines which express quantitatively different levels of ERBB1,-2, and -3 and ERBB1,-2,-3,and -4, respectively. Low dose radiation (2-4Gy) or 10ng/ml of a growth factor were used to stimulate wild type ERBB RTK phosphorylation and downstream signaling. For EGFRvIII, which is only expressed in vivo in tumor cells, co-expression with EGFR-CD533 was achieved through double adenoviral transductions.
175
I. J. Radiation Oncology
1027
● Biology ● Physics
Volume 54, Number 2, Supplement, 2002
Transducing Tat-Anti-MDM2 Peptides Enhances Cytotoxic Effect of Radiation and Chemotherapy on Hepatoma Cells IN VITRO
J. Cho, Y. Du, H. Dai, K. Chao Department of Radiation Oncology, Washington University, Saint Louis, MO Purpose/Objective: MDM-2, a negative regulator p53 tumor suppressor protein, is a potential target for developing novel molecular-targeted single and combined modality therapy for tumors expressing MDM2 and wild type p53. Hepatocellular carcinoma is known to be resistant to existing therapeutic approaches and therefore the prognosis remains poor. Here we examined the efficacy of a synthetic transducing Tat-␣MDM2 peptide and its combination with radiation and/or chemotherapy on the killing of hepatoma cells. Materials/Methods: We first assessed the biodistribution of Tat-peptide in vitro and in mouse organs. Cellular uptake and localization of Tat conjugated peptides were assessed using FITC labeled peptides after intra-peritoneal and intra-venous injection. Mouse hepatoma cell line (Hepa1c1c7) and mouse normal hepatocytes (BNL-CL2), both expressing wild-type p53, were used. The 12-aa MDM-2 binding domain of mouse p53 protein (QETFSGLWKLLP) was fused with a 12-aa transducing domain of HIV Tat protein at N-terminal during synthesis to create Tat-␣MDM2. Tat-peptide alone or a mutant form of the Tat-␣MDM2 peptide, which has the three MDM-2 interacting residues changed to Alanine, was served as the controls. Cell viability was quantified by clonogenic assay, colorimetric microplate MTS assay, or trypan blue exclusion test. Apototic cell death was confirmed by a colorimetric caspase-3 assay with CaspACETM using Ac-DEVE-p-nitroanilide as substrate and DNA breakage by TUNEL assay using fluorseine ApopTag威. Results: In cultured mouse and rat cells, cellular fluorescence peaks within 25 min after the addition of FITC peptides, with significant accumulation in both nuclei and cytoplasma. The distribution of FITC-Tat was assessed in vivo in nude mice bearing tumor xenografts on either flank. One hour after intra-peritoneal and intra-venous injection, substantial amount of FITC was detected in the subcutaneous tumors. Distribution to mouse liver is the most significant one among organs examined. This implicates the potential capability of this transducing Tat-Peptide in reaching tumor cells residing within the liver. We then turned to examine tumor-killing efficacy of Tat-␣MDM2 on hepatoma cells. The IC50 and IC90 for Tat-␣MDM2 were 300 mM and 500 mM, respectively for Hepa1c1c7. Tat alone or Tat-␣MDM2-mutant did not induce Hepa1c1c7 cell death at concentration up to 500 mM. No toxicity was observed for BNL-C2 cells with all peptides tested at up to 500 mM. Pretreatment with 150 mM Tat-␣MDM2, which did not produce detectable cell death when given alone, reduced the percent survival from 80% to 37% for Hepa1c1c7 cells irradiated at single dose of 5Gy. Similarly, pretreating Hepa1c1c7 with 150 mM Tat-␣MDM2 also lowered the IC50 for cisplatin from 25 mM to 11 mM. A 3.3- to 4.8-fold induction of p53 reporter luciferase activity was detected by Tat-␣MDM2 treatment in Hepa1c1c7 cells. Hepatoma cells treated with Tat-␣MDM2 stained strongly positive in TUNEL assay; however, Z-VAD-fmk did not protect Hepa1c1c7 cells against the killing effect of Tat-␣MDM2. Further, no caspase-3 activity was detected in cells treated with 500 mM Tat-␣MDM2, in contrast to 17 pmol pNA/mg/hour in those treated with 2 mM camptothecin. One possible factor that may have obscured the investigation of down-stream cell death paradigm is the rapid death of Hepa1c1c7. By trypan blue dye exclusion assay, percent cell death was 50%, 80%, and 99% at 30 min, 1 hour, and 2 hour, respectively, after 500 mM Tat-␣MDM2 treatment. Conclusions: Transducing Tat-peptide is capable of reaching cells in the liver after intra-peritoneal and intra-venous injection. We have shown that Tat-␣MDM2 peptide was selectively cytotoxic to hepatoma cells, and sublethal dose of Tat-␣MDM2 enhanced the cytotoxic effect of ionizing radiation and chemotherapy agent (cisplatin). With these encouraging in vitro results, we are proceeding with In Vivo investigation.
1028
Survivin Increases Resistance to Radiation-Induced Cell Death and is Upregulated by AKT Signaling in Primary Human Glioblastoma Cell Lines
A. Chakravarti, A. Chaklader, D.E. Latham, M.A. Delaney, J.S. Loeffler Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, Boston, MA Purpose/Objective: Survivin is a novel antiapoptotic gene that has been recently cloned and characterized. Its expression has been found to be of prognostic value in several tumor types, including human gliomas. Although Survivin has been found to suppress apoptotic cell death, its ability to inhibit radiation-induced cell death has been less well characterized. The purpose of this study was to investigate whether Survivin has a role in inhibiting radiation-induced cell death and, if so, to further investigate the underlying mechanism(s) involved. Materials/Methods: Two primary, tumor-derived, glioblastoma (GBM) cell lines were established in vitro and were both found to express Survivin by Western blot and by RT-PCR analysis. Inactivation of Survivin was accomplished in multiple ways: using antisense oligonucleotides, transient transfection experiments using dominant-negative constructs (nonphorylatable Thr(34)- Ala mutant called T34A), and by adenoviral vectors containing dominant-negative Survivin (Adeno-T34A). Subsequently, the cells were irradiated in doses ranging from 2 to 10 Gy, and survival was assessed using time-course apoptosis (Annexin V and cleaved caspase 3), viability, and clonogenic survival assays. As it was previously reported that AKT can upregulate Survivin levels in certain cell types, the AKT-Survivin relationship was also examined. Results: Inactivation of Survivin by any of these three methods significantly enhanced peak apoptosis values following radiation (P⬍0.0001) and reduced tumor cell viability and clonogencity (p⬍0.0001). Using selective pharmacologic inhibitors of caspase 3, it was determined that the reduction of the latter was, indeed, due to suppression of apoptosis. Interestingly, pharmacologic inhibition of caspase 8 reversed the enhanced cell death observed upon inactivation of Survivin, suggesting that Survivin can suppress both mitochondria-dependent and -independent apoptotic pathways. As would be expected, cultures of normal astrocytes failed to express Survivin, and Survivin inactivation failed to produce any demonstrable effects. Transient transfection experiments of GBM cell lines with dominant-negative, or kinase-deficient, AKT (AKT-KD), and with the constitutively active, or myristylated, form of AKT (AKT-Myr) suggest that AKT activity may be important in mediating accumulation of Survivin in the G1/S, as well as its known accumulation in the G2/M phases of the cell cycle.
Proceedings of the 44th Annual ASTRO Meeting
Conclusions: Survivin appears to enhance survival of GBM cells following radiation by suppressing both mitochondriadependent and -independent apoptotic pathways. Activation of AKT signaling appears to lead to accumulation of Survivin in the G1/S phases of the cell cycle, where it is normally absent. Given its exclusive expression in tumor cells, molecular targeting of Survivin appears to be a promising strategy to enhance the therapeutic gain of radiation in gliomas.
1029
Methylation of the Novel Apoptotic Gene TMS1/ASC in Human Glioblastoma Multiforme
1
W. Bobo , A. Stone1, D. Brat2, S.N. Devi3, E. Van Meir3, P.M. Vertino1 1 Department of Radiation Oncology, Emory University, Atlanta, GA, 2Department of Pathology, Emory University, Atlanta, GA, 3Department of Neurosurgery, Emory University, Atlanta, GA Purpose/Objective: Glioblastoma multiforme (GBM) is the most malignant of human brain tumors. Patients exhibit a mean survival of less than 12 months despite continued progress in neurosurgical techniques and the use adjuvant chemo- and radiation therapy. Defining the molecular events that contribute to the inititation and progression of these tumors, and an understanding of how these molecular events affect response to therapy are of critical importance in the tailoring of patient-specific treatment regimens. Acquired genetic and epigenetic alterations that lead to defects in apoptosis are common in human cancers, and promote tumorigenesis by allowing cells to survive under conditions that would normally trigger a cell death response. Unlike normal cells, cancer cells survive despite excessive DNA damage, in the absence of otherwise required growth factors, and under the hypoxic conditions that arise when a tumor outgrows its blood supply. Importantly, the propensity of tumor cells to undergo apoptosis is a critical determinant of their sensitivity to most chemotherapeutic agents and radiation. Therefore, defects in apoptotic signaling contribute to tumor initiation and progression, and can lead to drug resistance and treatment failure. Recently, we identified a novel gene, TMS1, that is silenced in association with aberrant DNA methylation in human breast and other cancers. TMS1 functions in the promotion of apoptosis, and acts upstream of caspase-9, a critical mediator of the apoptotic response to anticancer agents and other forms of cellular stress. Therefore, loss of TMS1 via aberrant methylation may contribute to human tumor development by allowing cells to bypass apoptosis and, as a consequence, may cause tumor cells to be resistant to anticancer drugs and radiation. The goals of the present study were to determine whether the TMS1 gene was aberrantly methylated in human glioblastomas, and to determine the relationship between TMS1 methylation and clinical disease course. Materials/Methods: Twenty-six human glioblastoma cell lines and twenty-five primary glioblastomas were evaluated for methylation of the TMS1 promoter using methylation-specific PCR. Tumor specimens were frozen immediately following surgical resection. All specimens were reviewed by a single neuropathologist for histologic confirmation of GBM and tissue integrity. Normal brain biopsies were obtained at autopsy from five cancer-free individuals. Expression of TMS1 was determined by reverse-transcriptase PCR. Results: We found that whereas normal brain tissue (5/5) was completely unmethylated at the TMS1 gene and expressed TMS1 mRNA, 10 of 25 (40%) of primary glioblastomas exhibited aberrant hypermethylation of the gene. In one case, there was complete methylation of the TMS1 gene. There was no correlation between methylation of TMS1 and patient age or sex. Interestingly, methylation of TMS1 tended to correlate with shorter post-operative survival times. Patients with tumors exhibiting an unmethylated TMS1 gene had a mean survival of 22 months (median⫽14) whereas those exhibiting aberrant methylation of the TMS1 gene had a mean survival of 9.6 months (median ⫽ 8). Conclusions: We are currently examining whether methylation of TMS1 may identify a subset of patients with reduced response to adjuvant chemo- or radiation therapy. Such findings would be consistent with the hypothesis that methylation of TMS1 confers resistance to cell death induced by chemotherapeutic agents and radiation, and would support the use of TMS1 methylation prognostic indicator for the treatment of glioblastoma.
1030
EGFR-CD533 Acts as a Pan-ERBB Receptor Tyrosine Kinase Inhibitor
J.N. Contessa, A. Abell, G. Lammering, T. Hewit, K. Valerie, R.K. Schmidt-Ullrich Department of Radiation Oncology, The Medical College of Virginia / Virginia Commonwealth University, Richmond, VA Purpose/Objective: The ERBB receptor tyrosine kinases (RTK), ERBB1 (EGFR), ERBB2, -3 and -4 are expressed at varied levels in human carcinoma and malignant glioma cells. These wild-type receptors as well as EGFRvIII, a constitutively active EGFR mutant lacking the extracellular N-terminal EGF binding domain, are involved in autocrine growth regulation of neoplastic cells. We have demonstrated that radiation induces indiscriminate activation of all ERBB species expressed by a given tumor cell which leads to powerful signals along the cytoprotective, mitogen-activated protein kinase (MAPK) pathway and confers radioresistance. Because of the nature of radiation-induced ERBB RTK activation only a pan-ERBB inhibitor can be expected to produce maximum radiosensitization. We have therefore investigated the effects of a dominant negative EGFR in blocking ERBB RTK signaling. Materials/Methods: The pan-ERBB inhibitory function of dominant negative EGFR-CD533, which lacks 533 C-terminal amino acids including the tyrosine kinase domain, is based upon the truncated receptor’s ability to interfere with ERBB RTK dimerizations and transactivation. We tested the effectiveness of the EGFR-CD533 in disrupting both growth factor and radiation-induced cytoprotective signals and its ability to confer radiosensitization using carcinoma and malignant glioma cell lines. Because of the ability to transduce human carcinomas with high efficiency and minimum toxicity, we employed an adenoviral vector, Ad-EGFR-CD533. The inhibitory effectiveness of EGFR-CD533 was studied in MDA-MB-231 and T47D breast carcinoma cell lines which express quantitatively different levels of ERBB1,-2, and -3 and ERBB1,-2,-3,and -4, respectively. Low dose radiation (2-4Gy) or 10ng/ml of a growth factor were used to stimulate wild type ERBB RTK phosphorylation and downstream signaling. For EGFRvIII, which is only expressed in vivo in tumor cells, co-expression with EGFR-CD533 was achieved through double adenoviral transductions.
175