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764. Harnessing PKR for the Selective Killing of Leukemia Cells Reveals Repression of the PKR Signaling Pathway
CANCER TARGETED GENE THERAPY II 763. The Antitumor Role of Chemokines Identified by SAGE Analysis in Murine Model Rejected GM-CSF-Transduced Tumor Cells: Implication of Novel Immune Gene Therapy Yoshihito Beppu,1,3 Hidenori Hase,2 Yuko Nakazaki,1 Kenzaburo Tani.1 1 Molecular and Clinical Genetics, Medical Institute of Bioreguration, Kyushu University, Beppu, Oita, Japan; 2 Immunolgy, Institute for Medical Science, Dokkyo University School of Medicine, Mibu, Tochigi, Japan; 3Japan Health Sciences Foundation, Chuo-ku, Tokyo, Japan. We previously reported that s.c. injection of WEHI-3B cells (5x10 5) transduced with GM-CSF resulted in the abolition of the ability to form solid tumor in vivo( Hase et al. ASGT 2000). To identify the molecules involved in the induction of the antitumor effects in this model, we analyzed the gene expression of the established tumor masses at 10 days after the transplantation by SAGE method and detected high expression of genes including chemokines. We particularly focused on chemokine genes of TARC and RANTES, which are expressed in dendritic cells, transduced WEHI-3B cells with each gene using retroviral vector (PMx neo) and investigated the antitumor effects of these chemokines in vivo. Mice injected subcutaneously with TARC or RANTES-secreting WEHI-3B(1x106) were found to have the growth of tumor inhibited. Mice injected subcutaneously with TARC or RANTES-secreting WEHI-3B cells (1x10 6) cotransduced with GM-CSF were inhibited strongly to have the tumor growth compared with those injected with WEHI-3B cells transduced only with GM-CSF, showing failure of forming solid tumor in 80% and 50% of injected mice respectively. These results suggest that TARC and RANTES are molecules strongly associated with antitumor effect of this murine tumor model and expected to become candidate genes for new generation immune gene therapy as the combination with GM-CSF gene.
764. Harnessing PKR for the Selective Killing of Leukemia Cells Reveals Repression of the PKR Signaling Pathway Inbar Friedrich,1 Hannah Ben-Bassat,2 Alexander Levitzki.1 1 Biological Chemistry, The Alexander Silberman Institute of Life Science, the Hebrew University of Jerusalem, Jerusalem, Israel; 2 Laboratory of Experimental Surgery, Hadassah University Hospital, Jerusalem, Israel. The double stranded (ds)RNA-dependent protein kinase, PKR, is a potent growth inhibitory protein that is most frequently activated in virally infected cells, inducing them to die. Selective activation of PKR in cancer cells could prove a powerful means of treating cancer. Many tumor cells express mutated genes containing deletions or chromosomal translocations that produce unique DNA sequences, and therefore unique RNA molecules. Antisense RNA, complementary to fragments flanking the deletion/ translocation, can produce a dsRNA molecule of sufficient length to activate PKR and induce cell death, upon hybridization with mutated, but not wild type mRNA. This hypothesis was successfully validated in our laboratory by utilizing a glioblastoma cell line, which expresses a truncated form of EGFR mRNA (Shir and Levitzki, Nature Biotechnology 2002). We attempted to apply this approach for selective killing of T-cell lymphoma cells that express the NPM/ ALK fusion. High infection efficiency was achieved by adenoviral infection of the T-cells. Antisense RNA was transcribed from novel adenoviral vectors containing the powerful U6 promoter, but antisense infection did not result in cell death. An in vitro, nonradioactive PKR kinase assay was developed in order to examine PKR activity in this cell line. Here we show that in this T-cell lymphoma cell line the endogenous eIF2α is not phosphorylated Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
upon activation of PKR by dsRNA, although the residue that is subject to phosphorylation (Ser51) is present. Furthermore, eIF2α in this cell line is phosphorylated on Ser51 following treatment with other stress agents. We are currently testing whether other cellular factors, such as p67, repress the PKR pathway in our cells. P67 is known to prevent phosphorylation of eIF2α, and we have found that p67 is expressed at high levels in this T-cell lymphoma.
765. Effect of STI571 Withdrawal and p210 Over-Expression on Tumorigenicity and Cell Viability of STI571-Resistant Chronic Myeloid Leukemia Colin L. Sweeney,1 Joel L. Frandsen,1 Catherine M. Verfaillie,2 Junia V. Melo,3 R. Scott McIvor.1 1 Gene Therapy Program, Institute of Human Genetics, Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN; 2Stem Cell Institute, Department of Medicine, University of Minnesota, Minneapolis, MN; 3Department of Haematology, Imperial College School of Science, Technology and Medicine, Hammersmith Hospital, London, United Kingdom. Chronic myeloid leukemia (CML) is characterized by the bcr-abl oncogene. STI571 is a potent inhibitor of p210bcr-abl tyrosine kinase, and has been very effective in clinical trials for treatment of chronicphase CML. However, STI571 has proven less effective for blastphase CML and bcr-abl+ acute lymphoid leukemia, with STI571resistant leukemia observed in some patients. Mutations within the kinase domain of bcr-abl and bcr-abl gene amplification have been implicated as mechanisms of STI571-resistance. We have been testing a gene therapy approach for treatment of CML involving marrow transduction with a retroviral vector expressing an antisense RNA directed against the bcr-abl message. This antisense has previously been tested in STI571-sensitive bcr-abl+ 32Dp210 and MO7e-p210 cell lines, and has been found to specifically reduce bcr-abl mRNA and p210 protein levels and decrease tumorigenicity. Antisense RNA might also be effective for treatment of STI571-resistant leukemia. The antisense is being tested in STI571-resistant murine Baf/BCRABL-r1 cells in which STI571-resistance results from increased p210 expression. These cells were cultured continuously in the presence of STI571 in vitro to maintain selective pressure for increased p210 expression. Further, the cells appeared to be dependent upon STI571 for growth in vitro, as increased cell death and decreased proliferation were observed short-term after STI571 was withdrawn. We found that transduction of Baf/BCR-ABL-r1 cells with the antisense vector did not reduce p210 protein levels or restore cell sensitivity to STI571, likely due to the high level of bcrabl over-expression as well as the selective pressure of maintaining cells in the presence of STI571. Further, we observed that Baf/ BCR-ABL-r1 cells did not cause tumors in Balb/c mice, unlike the parental STI571-sensitive Baf/BCR-ABL-s cells. To explain the lack of tumor formation observed when Baf/BCR-ABL-r1 cells were removed from STI571, we studied the effects of STI571 withdrawal on these cells. In apoptosis assays the cells exhibited increased apoptosis in the first 4 days after STI571 was withdrawn. However, the level of apoptosis (<10% apoptotic cells) did not correlate with the ≥1000-fold reduction in tumorigenicity. When Baf/BCR-ABLr1 cells were allowed to recover from STI571-withdrawal for 2 weeks in vitro, the cells were subsequently found to cause tumors, although with decreased potency compared to the STI571-sensitive Baf/BCR-ABL-s cells. Molecular analysis of specific apoptotic and proliferative signaling pathways and the level of p210 expression in the various cell populations is being conducted to further characterize the effects of STI571 withdrawal. We conclude that increased p210 expression was surprisingly associated with reduced tumorigenicity in STI571-resistant Baf/BCR-ABL cells, rendering this model S295
764. Harnessing PKR for the Selective Killing of Leukemia Cells Reveals Repression of the PKR Signaling Pathway Inbar Friedrich,1 Hannah Ben-Bassat,2 Alexander Levitzki.1 1 Biological Chemistry, The Alexander Silberman Institute of Life Science, the Hebrew University of Jerusalem, Jerusalem, Israel; 2 Laboratory of Experimental Surgery, Hadassah University Hospital, Jerusalem, Israel. The double stranded (ds)RNA-dependent protein kinase, PKR, is a potent growth inhibitory protein that is most frequently activated in virally infected cells, inducing them to die. Selective activation of PKR in cancer cells could prove a powerful means of treating cancer. Many tumor cells express mutated genes containing deletions or chromosomal translocations that produce unique DNA sequences, and therefore unique RNA molecules. Antisense RNA, complementary to fragments flanking the deletion/ translocation, can produce a dsRNA molecule of sufficient length to activate PKR and induce cell death, upon hybridization with mutated, but not wild type mRNA. This hypothesis was successfully validated in our laboratory by utilizing a glioblastoma cell line, which expresses a truncated form of EGFR mRNA (Shir and Levitzki, Nature Biotechnology 2002). We attempted to apply this approach for selective killing of T-cell lymphoma cells that express the NPM/ ALK fusion. High infection efficiency was achieved by adenoviral infection of the T-cells. Antisense RNA was transcribed from novel adenoviral vectors containing the powerful U6 promoter, but antisense infection did not result in cell death. An in vitro, nonradioactive PKR kinase assay was developed in order to examine PKR activity in this cell line. Here we show that in this T-cell lymphoma cell line the endogenous eIF2α is not phosphorylated Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
upon activation of PKR by dsRNA, although the residue that is subject to phosphorylation (Ser51) is present. Furthermore, eIF2α in this cell line is phosphorylated on Ser51 following treatment with other stress agents. We are currently testing whether other cellular factors, such as p67, repress the PKR pathway in our cells. P67 is known to prevent phosphorylation of eIF2α, and we have found that p67 is expressed at high levels in this T-cell lymphoma.
765. Effect of STI571 Withdrawal and p210 Over-Expression on Tumorigenicity and Cell Viability of STI571-Resistant Chronic Myeloid Leukemia Colin L. Sweeney,1 Joel L. Frandsen,1 Catherine M. Verfaillie,2 Junia V. Melo,3 R. Scott McIvor.1 1 Gene Therapy Program, Institute of Human Genetics, Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN; 2Stem Cell Institute, Department of Medicine, University of Minnesota, Minneapolis, MN; 3Department of Haematology, Imperial College School of Science, Technology and Medicine, Hammersmith Hospital, London, United Kingdom. Chronic myeloid leukemia (CML) is characterized by the bcr-abl oncogene. STI571 is a potent inhibitor of p210bcr-abl tyrosine kinase, and has been very effective in clinical trials for treatment of chronicphase CML. However, STI571 has proven less effective for blastphase CML and bcr-abl+ acute lymphoid leukemia, with STI571resistant leukemia observed in some patients. Mutations within the kinase domain of bcr-abl and bcr-abl gene amplification have been implicated as mechanisms of STI571-resistance. We have been testing a gene therapy approach for treatment of CML involving marrow transduction with a retroviral vector expressing an antisense RNA directed against the bcr-abl message. This antisense has previously been tested in STI571-sensitive bcr-abl+ 32Dp210 and MO7e-p210 cell lines, and has been found to specifically reduce bcr-abl mRNA and p210 protein levels and decrease tumorigenicity. Antisense RNA might also be effective for treatment of STI571-resistant leukemia. The antisense is being tested in STI571-resistant murine Baf/BCRABL-r1 cells in which STI571-resistance results from increased p210 expression. These cells were cultured continuously in the presence of STI571 in vitro to maintain selective pressure for increased p210 expression. Further, the cells appeared to be dependent upon STI571 for growth in vitro, as increased cell death and decreased proliferation were observed short-term after STI571 was withdrawn. We found that transduction of Baf/BCR-ABL-r1 cells with the antisense vector did not reduce p210 protein levels or restore cell sensitivity to STI571, likely due to the high level of bcrabl over-expression as well as the selective pressure of maintaining cells in the presence of STI571. Further, we observed that Baf/ BCR-ABL-r1 cells did not cause tumors in Balb/c mice, unlike the parental STI571-sensitive Baf/BCR-ABL-s cells. To explain the lack of tumor formation observed when Baf/BCR-ABL-r1 cells were removed from STI571, we studied the effects of STI571 withdrawal on these cells. In apoptosis assays the cells exhibited increased apoptosis in the first 4 days after STI571 was withdrawn. However, the level of apoptosis (<10% apoptotic cells) did not correlate with the ≥1000-fold reduction in tumorigenicity. When Baf/BCR-ABLr1 cells were allowed to recover from STI571-withdrawal for 2 weeks in vitro, the cells were subsequently found to cause tumors, although with decreased potency compared to the STI571-sensitive Baf/BCR-ABL-s cells. Molecular analysis of specific apoptotic and proliferative signaling pathways and the level of p210 expression in the various cell populations is being conducted to further characterize the effects of STI571 withdrawal. We conclude that increased p210 expression was surprisingly associated with reduced tumorigenicity in STI571-resistant Baf/BCR-ABL cells, rendering this model S295