ABSTRACT
Recent data suggest that tumor cells contaminating reinfused bone marrow may contribute to relapse in patients undergoing autologous bone marrow transplantation. Purging strategies that are able to remove these contaminating tumor cells need to be developed. This study describes how electroporation (EP) can be used to improve intracellular delivery of synthetic antisense oligodeoxynucleotides (ODNs), thereby enhancing their ability to suppress a target protein. Antisense ODNs that were introduced into cells by EP led to immediate suppression of targeted c-myc protein; this was associated with rapid cell death in the diffuse histiocytic lymphoma, U937; Burkitt's lymphoma, ST486; breast carcinoma, MCF-7; and Ewing's sarcoma, CHP-100, cell lines. Electroporation was found to have little or no detrimental effect on cells responsible for murine hematopoietic long-term reconstitution as determined from in vivo competitive repopulation studies. Using human c-myc-directed antisense ODNs as a model for the application of this approach to bone marrow purging, selective killing of human lymphoma U937 cells relative to normal human bone marrow cells was shown in cell mixing studies. In vivo studies were performed in which a survival advantage was shown for athymic mice that were inoculated with antisense-treated U937 cells as opposed to control cells. These studies suggest that EP of bone marrow may be of use in enhancing intracellular delivery of a variety of molecular/pharmaceutical agents. Taken together, these data suggest that the use of electroporation to enhance delivery of antisense ODNs is a promising new approach towards ex vivo bone marrow purging.
Subject(s)
Bone Marrow Purging/methods , Bone Marrow/pathology , Electroporation , Neoplastic Stem Cells/drug effects , Oligonucleotides, Antisense/pharmacology , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Adenocarcinoma/pathology , Animals , Apoptosis , Bone Neoplasms/pathology , Breast Neoplasms/pathology , Burkitt Lymphoma/pathology , Colony-Forming Units Assay , Female , Hematopoiesis/drug effects , Hematopoietic Stem Cells/drug effects , Humans , Lymphoma, Large B-Cell, Diffuse/pathology , Mice , Mice, Inbred C57BL , Mice, Nude , Neoplasm Transplantation , Oligonucleotides, Antisense/chemical synthesis , Proto-Oncogene Proteins c-myc/genetics , Sarcoma, Ewing/pathology , Tumor Cells, CulturedABSTRACT
These studies investigated the effectiveness of in vivo administration of cytokines in ameliorating potential marrow damage induced by chemotherapy. Breast cancer patients received 5-fluorouracil, leucovorin, doxorubicin and cyclophosphamide (FLAC) followed by either GM-CSF, PIXY321, or no cytokine. Marrow was obtained before and after one or two cycles of FLAC once blood cell counts had recovered. Colony-forming units for granulocytes and macrophages (CFU-GM) were used to indicate the effect of therapy on recovery of committed progenitor cells responsible for early blood cell recovery. The frequency and number of CFU-GM in marrow obtained after FLAC + PIXY321 were significantly lower than in marrow obtained after FLAC+GM-CSF or FLAC without cytokine. CD34+ cell numbers were also reduced after FLAC + PIXY321. CFU-GM production in marrow long-term cultures (LTC) was used to assess the effect of therapy on primitive progenitors. After 5 weeks the number of CFU-GM in LTC of post-therapy marrow from all three treatment arms was < 15% of the number in pre-therapy LTC. Suppressive effects of FLAC on primitive progenitors were observed even when committed progenitors and CD34+ cells had recovered to pre-therapy levels. These results demonstrate that cytokine treatment did not ameliorate suppressive or toxic effects of FLAC on the functional integrity of the marrow.
