INTERNATIONAL JOURNAL OF ONCOLOGY 11:281-287, 1997 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML Increased growth inhibition of human chronic myelogenous leukemic cells by a combination ofzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC c-myb antisense oligonucleotide and 4-hydroxyperoxycyclophosphamide in vitro ARVIN K.RAO 1 , CHARLES A. KUSZYNSKI 2 , ERIC BENNER 1 , PATRICK L. IVERSEN 3 , JOHN D. JACKSON 3 , MICHAEL R. BISHOP 4 and SHANTARAM S. JOSHI 1 Departments of Cell Biology and Anatomy, 2 Pathology and Microbiology, 3 Pharmacology, and internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198-6395, USA Received April 14, 1997; Accepted May 19, 1997 Abstract. Human chronic myelogenous leukemia (CML) is a unique malignancy in its cellular and molecular phenotypes. High dose therapy followed by stem cell transplantation seems to be one of the most effective treatment modalities for CML. However, allogeneic stem cell transplantation, a curative treatment modality, is limited due to the availability of matched siblings. On the other hand, the autologous stem cell harvests are contaminated with leukemic cells, and therefore a significant reduction of leukemic cells is desired before using the harvest for transplantation. Therefore in the present study, effects of a combination of a suboptimal concentration of 4-hydroxyperoxycyclophosphamide (4HC) and an optimal concentration ofzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA c-myb antisense oligo- nucleotide on the growth of K562 human chronic myelogenous leukemic cells in vitro were determined. The combination significantly (p<0.05) inhibited the growth of K562 cells in vitro when compared to the effects of c-myb oligonucleotide or 4HC alone. The c-myb oligonucleotide alone or in combination with low dose 4HC decreased the expression of c-myb gene as determined by RT-PCR techniques. Cellular uptake and retention of fluoresceinated oligonucleotide in control and treated K562 cells was studied using plain field laser microscopy and flow cytometry. There was an increase in cellular uptake of c-myb oligonucleotide in K562 cells as measured by plain field laser microscopy in the presence of 4HC. The combination of oligonucleotides and 4HC did not significantly decrease the number of hematopoietic stem/progenitor cells from normal hemato- poietic stem cell harvests as determined by in vitro colony assays. The combination of low dose 4HC and c-myb Correspondence to: Dr Shantaram S. Joshi, Department of Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198-6395, USA Key words: antisense oligonucleotide, c-myb, suboptimal dose of chemotherapy, synergistic effect, chronic myelogenous leukemia, growth inhibition antisense oligonucleotides can potentially be applied in CML patients, particularly for purging leukemic cells present in their hematopoietic stem cell harvests. Introduction Chronic myelogenous leukemia (CML) is a fatal disease when treated with only conventional chemotherapeutic agents. Though remission and cure can be obtained for some CML patients by allogeneic bone marrow, availability of suitable donor is a major problem. With autologous bone marrow, or peripheral blood stem cell transplantation, patients often relapse because of residual leukemic cells. Thus, a means to eliminate residual leukemic cells without an increase in systemic toxicity is needed. Antisense oligo- nucleotides, complementary to gene specific mRNA transcripts, can prevent the synthesis of a specific protein product by binding to its corresponding transcript in the cytoplasm of the cell (1,2). Tonkinson et al assert that specificity for the human genome is theoretically achieved by a statistically unique oligonucleotide sequence, consisting of 15-17 bases (2). Based on the results from preclinical and Phase I clinical trials which demonstrated a low or no toxicity to nomal tissues/organs, the prospect for antisense oligonucleotides as clinical therapeutic agents is encouraging (3). Antisense oligonucleotides can be targeted to specific genes that are crucial to the proliferation or survival of a tumor cell. Several such genes have already been identified and one such target gene is the c-myb gene which is the transcription factor involved in regulation of growth and differentiation of cells of the hematopoietic system (3-5). Several leukemias/lymphomas, including CML, are characterized by a translocation or overexpression of the c-myb gene (5). The antisense oligonucleotide complementary to c-myb mRNA has been used to inhibit tumor growth (4). Gewirtz reported leukemic cells to be more sensitive to c-myb proto-oncogene product deprivation than normal cells and suggested the c-myb oligonucleotide to be a sequence specific inhibitory agent of critical gene function and cell survival in vitro (6). Joshi et al reported the inhibition of in vitro growth of Raji, a human Burkitt's lymphoma cell line, by antisense oligonucleotides directed to c-myb mRNA (4).