188 I. J. Radiation Oncology l Biology l Physics Volume 48, Number 3, Supplement, 2000

Materials & Methods: The transcriptional cassette of the oriP element along with a basal CMV promoter was cloned in juxtaposition with either the bacterial B-galactosidase reporter gene or the p53 gene, and these were used to engineer two dElldE3 adenoviral vectors denoted as adv.oriPB-gal, or adv.oriPp53 respectively. Two NPC cell lines, the EBV-negative CNE-2Z, and EBV-positive C666-I, as well as the EBV-negative normal nasopharyngeal fibroblasts KS], were used in this study. In order to prove that adv.oriP-mediated B-gal expression was mediated by EBNAl, CNE-2Z cells were also stably transfected with EBNAl cDNA. Western blotting was used to evaluate p53 protein production, and the MTT assay was conducted to determine cell viability after gene transfer. The non-selective adv.CMV.B-gal or adv.CMVp53 were used to determine the extent of EBV-targeted expression by the newly constructed adv.oriP vectors.

Results: Infection of parent CNE-2Z, EBNAI-transfected CNE-2Z, C666-I, and KSI fibroblasts with adv.CMV.B-gal demonstrated significant expression in all cell lines. In contrast, infection of these 4 cell lines with adv.oriPB-gal demonstrated no expression in the KS 1 fibroblasts, < 1% X-gal staining in the parent CNE-2Z cells, and nearly 100% X-gal positivity in the EBNAI-transfected CNE-2Z and C666-1 cells. Western blot for p53 expression was conducted in C666-1 cells 48 hours after infection with adv.oriPp53, and significant p53 expression was observed. Results from the MTT assay for KSI and parent CNE-2Z cells after adv.CMVp53 (25 pfu/cell) infection demonstrated 20% and 60% cytotoxicity respectively. In contrast, only 5% and 15% cytotoxicity was observed in the same respective cell lines after infection with iso-effect doses of adv.oriPp53.

Conclusion: We have successfully developed a novel adenoviral vector, that incorporates an EBNA I -responsive element, and provides for selective transcription of a therapeutic gene in EBV-positive NPC cells. This vector will be evaluated further. in combination with other cytotoxic modalities, in order to maximize therapeutic gain in cancer gene therapy. The principle of this approach can be applied to all EBV-associated and other virus-associated human malignancies.

153 Th e a d enoviral p53 gene therapy enhanced the efficacy of radiotherapy in radioresistant prostate cancer cells

R. Sasaki,’ T. Shirakawa,‘.j A. Gotoh,’ Z. J. Zhan g,’ Y. Wada,’ A. Tamekane,’ K. Sugimura,’ M. Matsuo,’ S. Kamidono’

‘Kobe University School oj’Medicir?r, Dept. of Radiology, KOBE, Jupm, ‘Kobr Univrrsit.y School of Medicirtr, Dept. oj Urology, KOBE, Japan, ‘Kobr Utliveccity School of Medicine, Dept. of Intrrmtional Centrv for Mali& Research, KOBE, Japan, 4Kobe University School c?f Medicine. Dept. of Intrmal Medicine. KOBE, Japan

Purpose: In most androgen refractory or advanced prostate cancer, the incidence of mutations of the p53 tumor suppressor gene is remarkably increased. The wild-type p53 gene delivery itself has been shown to inhibit tumor cell growth. Moreover the p53 gene plays an important role in radiation-induced apoptotic cell death in various cancer cells. In this study, combination of ionizing radiation (IR) and adenoviral wild-type p53 gene therapy was investigated. Novel findings that adenoviral p53 gene therapy greatly enhanced cell growth inhibition by IR became apparent especially in radioresistant prostate cancer cells.

Materials & Methods:: The two human androgen refractory prostate cancer cell lines, DU145 and PC-3 cells, containing different types of p53 gene mutations, were investigated. The recombinant adenovirus vector containing wild-type p53 gene (Ad5CMV-p53) was used for this study. Cells were irradiated in tissue culture flasks (0. 2. 4, and 6 Gy, 3 Gy/min.) After I2 h. cells were infected with various doses of the Ad5CMVp53 (2.5 - 40 MOI). The cytotoxicity was determined by colony forming assay. The molecular mechanisms of this combination therapy were evaluated by quantitative RT-PCR (~53, bax and p21 mRNA expressions), histopathological apoptotic cell detection (Tune] assay), and cell cycle analysis by flow cytometry.

Results: 1) Significant cell deaths by IR were observed in a dose dependent manner in both cell lines. As DUl45 cells were less radiosensitive than PC-3 cells, cell growth inhibition in DU145 cells by IR was strongly enhanced by Ad5CMV-p53 infection in a viral dose dependent manner. 2) In DU145 cells, IR alone induced minimal bax and ~21, but not ~53, mRNA expression. However, IR combined with Ad5CMV-p53 infection stimulated significant increase in ~53. bax, and ~21 mRNA expression in DU145 cells. In PC-3, IR induced bax and p2l mRNA expression, while IR combined with Ad5CMV-p53 infection did not demonstrate significant change in these mRNA expressions. 3) Apoptotic cell deaths were rarely observed after IR alone (DU145: 3%, PC-3: 5%.) However, after combination therapy, the proportion of apoptotic cells increased seven-fold in DU145 cells, and twice in PC-3 cells. 4) GO/Cl cell cycle arrests were not seen after IR alone (DUl45: 30%, PC-3: 40%.) However, after the combination therapy. the GO/G1 arrest occurred only in DUl45 (DU145: 40%, PC-3: 15%.) 5) These findings about apoptotic cell deaths and cell cycle arrests were well correlated with the differences and the changes of bax and ~21 mRNA expressions.

Conclusion: In this study, we demonstrated that Ad5CMVp53 gene therapy could enhance the efficacy of IR especially in radioreaistant prostate cancer cells. These findings may pave the way for efficient radiation-gene therapy in various radiore- sistant cancers in the future.

154 Systemic gene therapy of HER-2/neu overexpressing breast cancer using radiation sensitizer ElA

.I. Y. Chang,’ R. Shao,’ W. Xia.’ S. Gupta-Burt,’ V. A. Saxena,’ M. C. Hung’

‘Rush-Presbyterian St. Luke Medical Crrtt<,r, Chicogo, IL, ‘MD Amleitrorz Cancer Cmter, Houstm. 7’X

Purpose: Overexpression of HER-2/neu haa been associated with poor clinic prognosis in breast cancer, which may be attributed partially to HER-2/neu induced radiation resistance. We reported previously that adenovirus type 5 ElA gene can function as a tumor suppressor by inhibiting HER-2/neu promoter activity. In animal models, we found that liposome-mediated local delivery of EIA can inhibit HER-2/neu expression and prolong mice survival in breast and ovarian cancer models. Phase I clinical trial of EIA gene therapy for patients with breast and ovarian cancer has been completed recently and the results appear very promising. In this study, we want to explore the possibility of using EIA as a systemic treatment agent and potential radiation sensitizer in HER-2/neu overexpressing tumor cells.

Materials and Methods: HER-2/neu-overexpressing cancer cell lines were transduced by either pElA plasmid or pEIA- mutant DNA in vitro. The stable transfectants were selected by adding G418. The cell growth rate and DNA fragmentation were

Proceedings of the 42nd Annual ASTRO Meeting IX9

analyzed after exposing to y radiation. The activity of the transcription factor nuclear factor-ltB (NF-KB) was detected by electrophoretic mobility-shift assays.

Orthotopic HER-2/neu overexpressing human breast cancer MDA-MB-361 nude mice model was treated with liposome EIA through tail vein. EIA gene expression and HER-2/neu down regulation in viva were detected by Western blot and immunochemical study. Tumor volume was monitored monthly. Biodistribution of liposome-mediated systemic gene transfer in viva was analyzed by liposome-pHClacZ-mediated IacZ gene transfer.

Similar approach will be applied to study the El A-mediated radiation sensitization in breast cancer nude mice model.

Results: To test whether EIA transduction can sensitize cancer cells to radiation, we tried to obtain stable transfectant by transducing ElA in HER-2/neu overexpressing breast cancer cell lines in vitro. However, EIA transduced HER-2/neu- overexpressing breast cells can not survival in vitro. Interestingly, EIA transduction of HER-2/neu overexpressing ovarian cancer cell line SKOV3ipl results in increased radiation sensitivity through inducing apoptosis that involves inhibition of the NF-KB.

Liposome-mediated systemic delivery of EIA in breast cancer mice model results in El A expression and HER-Z/neu down regulation in viva by 45% in western blot and 39% in immunochemical assay. The percentage of mice that continued to develop tumor in mice treated with PBS. a liposome-vector, and liposome EIA were lOO%, X0%, 60% respectively. More significantly, the tumor nodules from the El A-treated mice were much smaller than those in control mice, 134 2 X9, 101 2 105, 46.3 +- 53 in PBS-treated, liposome-vector-treated, liposome-EIA-treated mice respectively (n = IO, P 4 0.005). The biodistribution analysis showed a much stronger gene expression in tumor tissue compared with normal organs,

Combined systemic EIA gene therapy and radiotherapy in HER-2/neu overexpressing cancer models are undergoing.

Conclusion: Liposome-mediated systemic delivery of EIA can suppress HER-2/neu overexpresssion and achieve a therapeutic effect in viva. Preliminary data in vitro indicated that combined ElA gene therapy and radiation therapy may potentially achieve synergistic effect,

155 Analysis of a C-terminal truncation of the human Mrell protein: Implications for the molecular defect in ataxia telengiectasia related disorder (ATLD)

S. J. DiBiaae, L. M. Arthur, J. P. Carney

Utlirrrsity of Mot$and, Brdtinwt-e. MD

Purpose: The human Mrel l/RadSO/NbsI (MRN) protein complex is made up of three proteins, hMrel I, hRad50 and Nbsl This complex is intimately involved in the cellular response to DNA double-strand breaks. Recently, Stewart et al. (Cell 99577-587, 1999) have demonstrated that mutations of the hMREl1 gene are the causative mutations in two families that have the clinical characteristics of ataxia telangiectasia (ATLD). In order to understand the molecular defect of one of these mutations we have produced a mutant hMre1 I protein lacking the C-terminal 50 amino acids and examined its molecular properties.

Materials & Methods:: Recombinant wild-type and mutant Mrel I (mrellDC) proteins with 6X histidine tags were expressed using baculovirus in insect cells and puritied by Ni2+ affinity chromatography. The wild-type and mutant proteins were compared by ability to interact with hRad50 and Nbsl, DNA binding ability, and exonuclease assay.

Result: Using gel shift analysis and filter binding assay we demonstrate that the mrel IDC protein has a defect in its ability to bind to DNA. However, we see no defect in the ability of mrel IDC to interact with hRad50 and Nbsl or its ability to exonucleolytically degrade DNA.

Conclusion: The defect in DNA binding ability of the mrel 1DC protein may preclude the MRN complex from properly recognizing DNA double-strand breaks. Given that the MRN complex is involved in the detection of double-strand breaks this reduced binding affinity may explain the cellular phenotypes (i.e. radiation sensitivity and cell cycle checkpoint defects) observed in patients with a similar mutation in hMre1 I.

156 At’ n tsense inhibition of BCL-XL expression increases ionizing radiation (IR)-induced apoptosis in human lung carcinoma (Ca) cells and also alters the expression of other apoptosis-related gene products

H. Xiao. Y. Makeyev, N. M. Dean, B. Vikram, W. A. Franklin

‘Albert Einstein Collqr of Medicbw, Bronx. NY. 21SIS Pharmaceutics, Cnrlsbad, CA

Purpose: Pre-clinical and clinical studies suggest that Bcl-XL (a homologue of Bcl-2) is an important inhibitor of IR or chemotherapy-induced apoptosis in cancer cells, and that cancers expressing Bcl-XL carry a worse prognosis. Lung ca has a poor prognosis. an d the radiotherapy dose is limited by potential toxicity. A study with lung ca cells that were resistant to IR-induced apoptosis was undertaken to determine if inhibiting Bcl-XL expression would (1) increase IR-induced apoptosis and (2) alter the expression of other apoptosis-related genes.

Material & Methods: The Bcl-XL specific antisense oligonucleotide ISIS 16009 was designed to target Bcl-XL and was synthesized as a 20 gapmer. ISIS 16009 was used to inhibit Bcl-XL expression in a human lung adenocarcinoma cell line, A.549, and two derivative lines: E6, in which p53 function was disrupted by human papilomavirus E6. and LXSN, a transfection control line. The mRNA expression was determined using an RNA protection assay (RPA) and protein expression was measured by Western analysis. The percentage of apoptotic cells was evaluated by using both flow cytometry and staining with Hoechst 33258.

Results: ISIS 16009 inhibited Bcl-XL mRNA and protein expression, in a dose-dependent and sequence-specific manner, in each of the three cell lines. Radiation alone or ISIS 16009 alone produced minima1 apoptosis: however. the combination of the ISIS 16009 and g-radiation resulted in extensive apoptosis: in -35% of the A549 cells (~53 wild-type) and -45% of the E6 cells (~53 disrupted). A549 cells expressed Bcl-W. BID, Bak, Bax, and MCI-1, but not Bcl-2, Bfl-1 or Bik. However. when