1077 Radio Responsive Gene Transfection of Malignant Glioma Cells Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������®������������ �!����� ����"� �������� S415 � ����� ��� ���� tr[.]
CANCER APOPTOSIS transfected wild type E1A into these cells and then treated with paclitaxel (Taxol) in vitro and in vivo In the in vitro study, the E1Aexpressing MDA-MB-231 (231-E1A) and MCF-7 (MCF-7-E1A) cells were exposed to different doses of Taxol and the cellular cytotoxicities of Taxol were evaluated by MTT cytotoxicity assays, PARP cleavage, and FACS analysis The results showed that expression of E1A enhanced in vitro Taxol cytotoxicity, as compared to the control cells For the in vivo study, we first compared the therapeutic efficacy of Taxol between orthotopic tumor models established with parental MDA-MB-231 versus 231-E1A stable cells, using tumor weight and apoptotic rate (TUNEL assay) as the parameters We found Taxol was more effective in shrinking tumors and inducing apoptosis in tumor models established with stable 231-E1A cells than the control cells We then tested whether E1A could directly enhance Taxol-induced killing in nude mice, by designing a systemic E1A gene therapy experiment using intravenous (i.v.) injection of the E1A gene via the mouse tail vein We compared the therapeutic effects of E1A gene therapy with or without Taxol chemotherapy in the established orthotopic tumor model of animals inoculated with MDA-MB-231 cells, and found that a combination of systemic E1A gene therapy and Taxol chemotherapy significantly enhanced the therapeutic efficacy and dramatically repressed tumor growth (P < 0.01) In addition, survival rates were significantly higher in animals treated with combination therapy than in the therapeutic control groups Three of the seven animals treated with combination therapy achieved more than one year of tumor-free survival Our data showed that expression of E1A significantly enhanced both chemosensitivity and an anti-tumor effect induced by Taxol as well as prolonging animal survival rates in the orthopic in vivo model This work was supported in part by NIH Grant RO1-CA58880 and the SPORE grant for ovarian cancer research from the National Institutes of Health (to M.-C H.) and DAMD17-01-1-0300 from the United States Department of Defense Army Breast Cancer Research Program (to Y L.) 1075 Transcription Factors NF-kB and AP-1 as Targets for Prostate Cancer Gene Therapy Luiz F Zerbini,1 Yihong Wang,1 Jey Y Cho,1 Xuesong Gu,1 Jon Jones,1 Mehmet Inan,1 Charles Bailey,1 Marie Joseph,1 Jinrong Zhou,1 Towia A Libermann.1 Medicine, BIDMC Genomics Center/Harvard Medical School, Boston, MA It has been suggested that NF-kB and AP-1 may play a role in the survival of prostate cancer cells NF-kB activity in human prostate cancer cells has been associated with invasion and metastasis and enhanced TNF-a induced apoptosis Similarly, AP-1 activation in prostate cancer cell lines has been implicated in cell differentiation, proliferation, apoptosis and oncogenic transformation We have investigated whether interference with NF-kB and/or AP-1 activity interferes with prostate cancer growth and survival Our data show that the blockage of NF-kB and AP-1 transcription factors by an adenovirus encoding the IkB-alpha gene and the dominant-negative JunD, respectively, inhibits prostate cancer cell proliferation and induces cell death in DU145 cells The apoptosis in DU145 cells is due to upregulation of pro-apoptotic genes such as Fas and Fas ligand in a mRNA and protein levels as demonstrated by real time PCR, western blot and ELISA assays and was mediated via caspase and Additionally, overexpression of the IkB gene inhibits tumor growth and IL-6 expression after orthotopic implantation into the prostate of nude mice Furthermore, IkB overexpression leads to activation of MAP kinase pathways in DU145 prostate cancer cells as demonstrated by western blot analysis and kinase assays Using specific kinase inhibitors we demonstrate that MAP kinase pathways are important for apoptosis induction Microarray analysis of more Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy than 18000 human genes further delineates the apoptotic pathways regulated by IkB-alpha and the dominant-negative JunD These data show that NF-kB and AP-1 transcription factors play an important role in prostate cancer progression and apoptosis and the combined interference with these transcription factors may give rise to novel therapeutic modalities in the fight against prostate cancer 1076 DNA Chip Technology and Proteomics in Melanoma: Identification of New Targets for Gene Therapy Alireza Mirmohammadsadegh, Annett Baer, Klaus Werner Schulte, Walter Bardenheuer, Ulrich Remigius Hengge Dermatology, Heinrich-Heine-University, Duesseldorf, NRW, Germany One important application of DNA microarray technology is the simultaneous analysis of gene expression of different mRNA species Comparison of mRNA patterns of diseased and healthy tissue may help to understand the pathogenesis of a given disorder Identified dysregulated genes for example in cancer tissue may function as new molecular markers for diagnosis or prognosis or may ideally serve as a new target for therapy Using membrane cDNA arrays technology and 2D-gel electrophoresis and mass spectrometry we analysed gene and protein expression of human melanoma, one of the most agrgressive types of cancer with a high metastaic potential and with markedly increasing incidence worldwide To account for the heterogeneity of different tumors we collected total RNA from 10 different melanoma metastases from 10 different patients The RNA pattern of the pooled specimens was compared to gene expression of primary human melanocytes using different cDNA membrane arrays from Clontech® with appromximaly 1940 genes An abudance of genes were dysregulated (up/down) which involved for examlpe in apoptosis like FAST, TFAR 15, GRB10, or in angiogenesis like VEGF Here, we focus our description on the JAK/STAT signaling pathway which is involved in cell proliferation, cell differentiation and apoptosis Stat-induced inhibitor (SSI-2) is upregulated in human primary melanocytes in comparison to human malignant melanoma metastases SSI-2 is a member of a protein family, that inhibits cytokine responses and activation of ´signal transducer and activator of transcription´ (STAT) Therefore, we investigated the protein expression of Stat3 and Stat5 via Western blot analysis and observed a constitutive level of these proteins in primary melanocytes as well as in malignant melanoma metastasis In contrast, the phospho-Stat3 and phosphoStat5 was weakly expressed in primary melanocytes and upregulated in melanoma metastases The upregulation of SSI-2 in primary melanocytes seemed to regulate Stat3 and Stat5 activation in negative manner This leads to normal proliferation and differentation of melanocytes In contrast to this lack of SSI-2 and the upregulation of phospho-Stat3, phosph-Stat5 in melanoma metastases seem to be involved in dysregulation of proliferation Therefore, targeting of Stat3, (e.g by dominant negative Stat3β) and Stat5 signaling may provide a potential therapeutic strategy in malignant melanoma 1077 Radio-Responsive Gene Transfection of Malignant Glioma Cells H Tsurushima,1 X Yuan,2 L Dillehay,2 K W Leong.1 Department of Biomedical Engineering; 2Department of Oncology, Johns Hopkins School of Medicine, Baltimore, MD, United States Lesion-restricted gene therapy is an attractive approach to reduce overall toxicity in cancer treatment For instance, gene expression S415 CANCER APOPTOSIS can be localized to tumors or lesions, using tissue-type, or radiationsensitive promoter elements In this study we present the possibility of achieving targeted cancer gene therapy using the combination of caspase-3 gene transfer and radiation Caspase-3 is a member of the cysteine protease family that plays a critical role in inducing apoptosis We inserted the cDNA of caspase3, which was picked up with RT-PCR, into the multiple cloning region of pCI-neo vector that is driven by a CMV promoter (pCICSP3) We then replaced the CMV promoter with the early growth response gene-1 (Egr-1) promoter, which is responsive to radiation (Egr-CSP3) The pCI-GFP was used as the reporter gene These plasmids were transfected against U251 and U87 glioma cell lines with Lipofectamin2000 The cells were irradiated at hrs after addition of the transfection complexes The frequency of apoptosis of the glioma cells was measured by Annexin V binding assay and FACS The killing activity was quantified by the crystal violet (CV) assay The transfection rate with pCI-GFP was 70.3% and 68.5% for U251 and U87 cells, respectively, at ug plasmid dose per 105 seeded cells and without irradiation Neither significant cell death nor apoptosis on U251 and U87 glioma cells could be detected with caspase-3 gene transfection alone Radiation in addition to caspase-3 gene transfection, from to 15 Gy, would heighten cell killing and apoptosis significantly At ug DNA dose and 15 Gy irradiation, cell kill of U251 cells was increased from 19 to 60%, and the percentage of apoptotic surviving cells increased by 17% Similar magnitude of radiation enhancement in cell kill and apoptosis was observed for U87 cells Supplement of a caspase-3 or caspase-9 inhibitor would completely eradicate the radiation enhancement effect Transfection of the glioma cells with the Egr-CSP3 construct, which contains a radiation-sensitive promoter, produced similar effects as the pCI-CSP3 construct, in that transfection alone had no effect on cell kill and apoptosis, but was significantly augmented by radiation However, although containing a radiation-sensitive promoter, the Egr-SCP3 was not as effective as PCI-CSP3 in the radiation enhancement effect This is attributed to the fact that the Egr-1 promoter is considerably weaker than the CMV promoter and consequently could not produce sufficient transgene expression Given the localized nature of radiotherapy, this study suggests that caspase-3 gene therapy in conjunction with radiation might constitute an interesting strategy to achieve targeted cancer gene therapy 1078 Gene Therapy for Uterine Fibroids II: Adenovirus-Mediated Expression of Dominant Negative Estrogen Receptor Ablates Pre-Existing Leiomyomas in Nude Mice Ayman Al-Hendy,1 Eun J Lee,2 Hui Q Wang,3 John A Copland.4 Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX; 2Endocrinology, Northwestern University, Chicago, IL; 3Sealy Center, UTMB, Galveston, TX; 4Int Med, UTMB, Galveston, TX Introduction: Uterine leiomyomas (fibroids) are the most common tumors in premenopausal women Currently there is no medicinal treatment for this condition and surgery is the main stay This constitutes a clinical dilemma in fibroid patients who desire to preserve their fertility, who are not fit for surgery, or who are pregnant We have recently (J Soc Gyn Invst, 9:65A, 2002) demonstrated the ability of a mutated dominant-negative estrogen receptor gene delivered via an adenoviral vector (AdDN-ER) to induce apoptosis in human and rat leiomyoma cells Objective: To assess the ability of AdDN-ER to ablate preexisting fibroids when delivered by direct intratumor injection Methods: Female Balb/C nude mice were implanted subcutaneously (SC) with 5x106 ELT3 cells (rat leiomyoma cells) Three weeks post-implantation, all mice developed SC palpable S416 tumors and were randomized into three groups Group one was treated with AdDN-ER estimated at100 pfu/cell Group two treated with Ad-LacZ (marker gene), and group three with medium alone and served as controls All treatments were delivered by direct intratumor injection via four separate entries Mice were closely followed and tumor volume measured using a digital caliper Sample animals were sacrificed at regular intervals to characterize the proliferative (BrdU) and apoptotic (TUNEL) indices Results: The direct intratumor injection of adenovirus was uneventful and well tolerated by all mice The AdDN-ER-treated mice demonstrated immediate overall arrest of tumor growth with evident tumor regression in some mice During same time period, the tumors in the control groups continued to grow exponentially increasing their volume by 500%-800% within two weeks The difference in fibroids volume between AdDN-ER-treated mice and control groups was highly significant (P=0.007) The AdDN-ERtreated tumors demonstrated severely-inhibited cell proliferation (BrdU index = 4.4%±2) compared to control groups (52%±6, and 59%±8, P