Luketich,2 Joel S. Greenberger.1. 1Radiation Oncology, University of Pittsburgh Cancer Institute,. Pittsburgh, PA; 2Thoracic Surgery, University of Pittsburgh.
CANCER TUMOR SUPPRESSOR GENE AND APOPTOSIS 671. Overexpression of Manganese Superoxide Dismutase (MnSOD) Prevents Photodynamic Therapy (PDT)-Induced Porcine Esophageal Stricture 1
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Anurag Agarwal, Michael W. Epperly, Yaron Perry, James D. Luketich,2 Joel S. Greenberger.1 1 Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 2Thoracic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA. Photodynamic therapy (PDT) is used in treatment of esophageal cancer and for ablation of Barrett’s esophagus. A complication of this procedure is esophageal stricture. We sought to determine whether overexpression of MnSOD prevented formation of PDTinduced esophageal stricture in a pig model. Pigs were injected intravenously with Photofrin (2 mg/kg) and MnSOD-PL complex (10 mg plasmid DNA) by an endoscope placed either at the site of PDT treatment (10 cm from gastroesophageal (GE) junction) or at the top of the esophagus, which allowed the pig to swallow the plasmid/liposome complex thereby coating the entire esophagus. An endoscope was placed 10 cm from the GE junction after injection of Photofrin and MnSOD-PL, a laser was inserted through the endoscope, then 400 Joules of light was delivered to the esophagus. PDT treatment was repeated 48 hours later. Control pigs were treated as above with no injection of MnSOD-PL. Pigs were followed for the development of esophageal stricture by weekly endoscopic exam and were weighed every two days. Esophageal biopsies were collected at various times following PDT. RNA was extracted and quantitative real time PCR was performed to determine gene expression for inflammatory cytokines including TNF-a and TGFβ. At 14 days after PDT treatment, control pigs lost significant weight and esophageal stricture was detected by endoscopic exam. By 20 days after PDT treatment, control pigs had severe esophageal stricture determined by endoscopy, weight loss, and barium swallow. Esophageal stricture in control PDT-treated pigs was documented by histopathology. In contrast, pigs intraesophageally treated with MnSOD-PL 24 hours before PDT had no weight loss, showed continual weight gain through 150 days after PDT and no esophageal stricture was detected by endoscopic exam. At 150 days after treatment, all MnSOD-PL-treated pigs were sacrificed, esophagus examined, and no stricture was detected. Thus, increased expression of MnSOD-PL may protect normal esophagus from PDT-induced stricture. Joel S. Greenberger, M.D. is a member of the Scientific Advisory Board and is a holder of equity in Automated Cell, Inc., Pittsburgh, PA
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672. Lentiviral Transduction of INK4A/ARF Protein Cooperates with Anti-Leukemic Agents To Induce Apoptosis of Leukemia Cells Yuansong Bai,1 Yasushi Soda,1,2 Minghan Chen,1 Kiyoko Izawa,1 Seiichiro Kobayashi,1 Akira Tomonari,2 Jun Ooi,1,2 Fumitaka Nagamura,2 Satoshi Takahashi,2 Kaoru Uchimaru,2 Tohru Iseki,3 Hiroyuki Miyoshi,4 Tsuneo A. Takahashi,5 Kenzaburo Tani,1,6 Arinobu Tojo,1,2 Shigetaka Asano.1,2 1 Division of Molecular Therapy, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 2Department of Hematology/ Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 3Department of Transfusion Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 4Division of Cell Processing, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 5Subteam for Manipulation of Cell Fate, BioResource Center, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan; 6Department of Advanced Molecular and Cell Therapy, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Fukuoka, Japan. Inactivation of the INK4A/ARF locus has been implicated in tumorigenesis, particularly in leukemogenesis. It has been found in 25-60% cases of acute leukemia and 20-50% cases of lymphoma. Although deletion of the INK4A/ARF locus is uncommon in AML, mRNA expression is likely to be disrupted, resulting from hypermethylation or mutation in the promoter region. There is some evidence that restoration of p16INK4a into p16-deficient leukemia cell lines suppresses their proliferation in suspension culture or their clonogenic growth in semisolid culture. However, previous studies were conducted by retrovirus or plasmid-mediated gene transfer under drug selection, suggesting low efficiency of transduction which appears disadvantageous to interpret the results of the growth inhibitory gene. Here we constructed a lentiviral vector expressing either p16ink4a or p14arf respectively, and validated its effect on leukemia cells, especially in combination with anti-leukemia agents including imatinib, ATRA and TNF-α. The p16, p14, hrGFP or mock lentiviral transfer vector was introduced into 293T cells along with packaging plasmids (pMDLg/p.RRE and pMD.G) and pRSVRev. The resulting VSV-G-pseudotyped lentivirus supernatant was concentrated by ultracentrifugation for high-titer virus stock. K562, NB4 and IMS-M2 cells were infected with those prepared viruses at the multiplicity of infection (moi) of 20-50, which allowed almost 100% of infection efficiency based on hrGFP fluorescence. The former two cell lines were deficient in p16 and p14 transcripts and had mutated p53 but intact pRb, while the latter one expressed both transcripts and had mutated p53 but intact pRb. Overexpression of p16 induced leukemia cells to accumulate in the G1 phase, followed by partial (IMS-M2) or complete (K562 and NB4) inhibition of cell growth. In addition, overexpression of p14 resulted in partial (IMSM2) or substantial (K562 and NB4) apoptotic cell death, suggesting the presence of p53-independent pathway of apoptosis through p14. Next, we transduced p16 and p14 in primary blast cells from CML-BC or AML patients, resulting in their growth inhibition and apoptosis with a patient to patient variation. On the other hand, clonal growth and differentiation of cord blood progenitor cells was not affected by enforced expression of either p16 or p14 at the same moi. Next, not only imatinib and TNF-α-induced apoptosis of K562 and IMS-M2 cells but also ATRA-induced apoptosis of NB4 cells were markedly enhanced by transduction of p14 at a low moi less than 2.5. These results suggest that INK4A/ARF protein-mimetic agents may be promising options for leukemia in combination with the present anti-leukemia agents since these diseases are relatively resistant to a single agent.
Molecular Therapy Volume 9, Supplement 1, Ma y 2004
Copyright The American Society of Gene Therapy
