title: Osteoclasts direct bystander killing of cancer cells in vitro.
abstract: Cytosine deaminase (CD) catalyzes the deamination of 5-fluorocytosine (5FC) to produce the highly toxic chemotherapeutic agent 5-fluorouracil (5FU). A unique feature of the CD/5FC enzyme/prodrug system is its ability to kill adjacent cells via bystander killing. Bystander killing of cancer cells can be mediated by non-cancerous accessory cells transduced with the CD gene; one type of non-cancerous accessory cell found in primary bone cancer and breast cancer metastases to bone is the osteoclast. This manuscript determines if osteoclast precursor cells, transduced with the CD gene, can function as a gene delivery system capable of killing cancer cells. An osteoclast precursor cell line (RAW 264.7, RAW) and authentic bone marrow-derived osteoclast precursor cells were transduced with a retroviral vector containing the cytosine deaminase fusion gene (NCD) composed of the human nerve growth factor receptor and CD genes. RAW cells and bone marrow-derived osteoclast precursor cells transduced with NCD expressed NCD protein and converted 5FC to 5FU. Treatment of NCD-transduced osteoclast precursor cells with the 5FC prodrug resulted in significant killing in vitro. NCD-transduced osteoclasts were co-cultured with either DsRed2-labeled sarcoma cells (2472-DSR) or green fluorescent protein (GFP)-labeled breast cancer cells (GFP-4T1). Treatment of the NCD osteoclast/tumor cell co-cultures with 5FC resulted in bystander killing of 2472-DSR cells (P < 0.006) and GFP-4T1 cells (P < 0.004). These findings demonstrate that NCD-transduced osteoclasts can promote killing of cancer cells and introduce the exciting possibility for developing osteoclast-mediated, CD-based treatment of primary bone cancers and breast cancer metastases to bone.
authors.affiliation: Department of Orthopedic Surgery, University of Minnesota, 420 Delaware Street SE, MMC 806, Minneapolis, MN 55455, USA.
authors.forename: Margaret
authors.initials: M
authors.lastname: Ramnaraine
authors.forename: Weihong
authors.initials: W
authors.lastname: Pan
authors.forename: Denis R
authors.initials: DR
authors.lastname: Clohisy
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000633:toxicity; D001854:Bone Marrow Cells / Q000166:cytology; D001859:Bone Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D043525:Cytosine Deaminase / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D005437:Flucytosine / Q000378:metabolism / Q000633:toxicity; D005472:Fluorouracil / Q000633:toxicity; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008264:Macrophages / Q000166:cytology; D051379:Mice; D041681:NIH 3T3 Cells; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D020800:Receptor, Nerve Growth Factor / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012509:Sarcoma / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D014161:Transduction, Genetic
chemical_list: D000964:Antimetabolites, Antineoplastic; D020800:Receptor, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005437:Flucytosine; D043525:Cytosine Deaminase; D005472:Fluorouracil