Osteoclasts direct bystander killing of cancer cells in vitro
Introduction
One approach for cancer gene therapy involves using the cytosine deaminase gene (CD) to kill tumor cells [1], [2], [3], [4], [5], [6], [7]. The CD suicide gene catalyzes the deamination of non-toxic 5-fluorocytosine (5FC) to produce the highly toxic chemotherapeutic drug 5-fluorouracil (5FU). An interesting property of the CD enzyme/prodrug system is its bystander effect, which causes the death of unmodified tumor cells adjacent to genetically modified cells [8], [9]. Therapeutic efficacy of this approach relies on the bystander effect, where 5FU released from CD-expressing cells diffuses across tumor cell membranes. Recent reports indicate that CD gene delivery to tumors by non-cancerous accessory cells is a promising treatment for gliomas and lung cancer. The non-cancerous accessory cell chosen as a gene delivery system for gliomas is the neural progenitor cell [1], [10], and the cellular gene delivery system for lung cancers is the endothelial precursor cell [7], [11].
Recent advances in understanding the pathophysiology of bone cancer have provided keen insight into the pathologic role of osteoclasts [12], [13], [14]. It has been shown that these bone-resorbing cells form in high numbers at sites of bone cancer, destroy bone at sites of tumor, and stimulate tumor progression. As a first step toward developing a novel gene therapy for treating bone metastases, we hypothesized that CD-expressing osteoclast precursor cells can serve as a cellular gene delivery system capable of killing cancer cells.
To examine the possibility that osteoclasts can direct killing of cancer cells, we have transduced an osteoclast precursor cell line and authentic osteoclasts with a customized yeast CD gene. Findings indicate that CD-transduced osteoclast precursors form osteoclasts, express active CD, convert the prodrug 5FC to 5FU, and direct bystander killing when co-cultured with murine sarcoma or breast cancer cells. Findings represent a crucial first step toward proving the exciting possibility that CD-expressing osteoclast precursor cells can be used to develop new cancer gene therapy treatments for primary bone cancer and bone cancer metastases.
Section snippets
Cell culture
RAW 264.7, a monocyte/macrophage cell line which can be differentiated into osteoclasts [16], [17], 2472 cell line, originally derived from a malignant tissue tumor (sarcoma) in a C3H mouse, NCTC clone L929 (L929), a macrophage colony-stimulating factor (MCSF) producing tumor line, NIH 3T3 normal mouse fibroblasts, and PA317 packaging cell line were obtained from the American Type Culture Collection (Rockville, MD). The 4T1 murine breast cancer cell line was a gift from Dr. Fred Miller [18] and
Characterization of transduced osteoclast precursor (RAW 264.7) cell line
RAW cells transduced with LNGFR-CDSN vector expressed high levels of the NCD fusion protein. As expected, the parent RAW cells and transduced cells (RAW/NCD) were osteoclast precursors in that they expressed surface membrane CD11b (Fig. 1). In contrast to RAW cells, RAW/NCD cells expressed the NGFR and CD portions of the NCD fusion protein. FACS analysis revealed abundant cell surface NGFR in RAW/NCD cells and no NGFR expression in RAW cells (Fig. 1). Western analysis of RAW/NCD cell lysates,
Discussion
Findings from this investigation demonstrate that CD-transduced osteoclasts can direct killing of sarcoma and breast cancer cells. Authentic bone marrow-derived osteoclast precursors and the osteoclast precursor RAW cell line were both transduced with the LNGFR-CDSN retroviral vector. Under osteoclastogenic conditions, each population of transduced cells formed osteoclasts and each population expressed the therapeutic NCD fusion protein. Measures of CD enzyme activity determined that both
Acknowledgments
We would like to acknowledge the assistance of the Flow Cytometry Core Facility of the University of Minnesota Cancer Center, a comprehensive cancer center designated by the National Cancer Institute, supported in part by P30 CA77598. Support was also provided by the National Cancer Institute; CA90434 and the National Institute of Arthritis, Musculoskeletal and Skin Diseases; and the Roby C. Thompson Endowment.
References (32)
- et al.
The emerging fields of suicide gene therapy and virotherapy
Trends Mol. Med.
(2002) - et al.
Embryonic endothelial progenitor cells armed with a suicide gene target hypoxic lung metastases after intravenous delivery
Cancer Cells
(2004) - et al.
Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells
Cell
(1997) - et al.
Osteoprotegerin: a novel secreted protein involved in the regulation of bone density
Cell
(1997) - et al.
Analysis of distinct tartrate-resistant acid phosphatase promoter regions in transgenic mice
J. Biol. Chem.
(2005) - et al.
Transplantation of prodrug-converting neural progenitor cells for brain tumor therapy
Cancer Gene Ther.
(2003) - et al.
In vivo gene therapy for colon cancer using adenovirusmediated, transfer of the fusion gene cytosine deaminase and uracil phosphoribosyltransferase
Gene Ther.
(2001) - et al.
In vivo cancer Gene Therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene
Cancer Res.
(2000) - et al.
Gene directed enzyme/prodrug therapy of cancer: historical appraisal and future prospectives
J. Cell. Physiol.
(2001) - et al.
Direct and bystander killing of sarcomas by novel cytosine deaminase fusion gene
Can. Res.
(2003)