It will be intriguing to test these and other hypotheses directly in living animals by determining whether zoledronic acid induces the bioluminescence reporter that has been introduced transgenically or retrovirally into distinct host cell populations
It will be intriguing to test these and other hypotheses directly in living animals by determining whether zoledronic acid induces the bioluminescence reporter that has been introduced transgenically or retrovirally into distinct host cell populations. likely to exert anti-tumor activity indirectly by targeting host cells. Accordingly, these findings shift attention toward the goal of determining which host cell types are targeted directly by aminobisphosphonates to exert adjuvant chemotherapeutic activity. Introduction The mevalonate biosynthetic pathway operates in all human organs and cell types to provide precursors for synthesizing steroids and isoprenoids that maintain cell membrane structure, function as endocrine hormones, produce heme A and ubiquinone for electron transport, ITE or change proteins post-translationally with isoprenoid lipids (prenylation) or N-linked oligosaccharide chains(1). Mevalonate pathway inhibitors that blunt protein prenylation are being investigated intensively for treating malignancy and other diseases. For example, statins, which inhibit HMG-CoA reductase to treat hypercholesterolemia, are under investigation in malignancy and dementia(2-4). Aminobisphosphonates, which inhibit farnesylpyrophosphate synthase (FPP synthase) in osteoclasts to reduce bone loss in osteoporosis and metastatic malignancy(5-8), are being analyzed preclinically and clinically as adjuvant chemotherapeutics that exert antitumor effects in breast cancer(9-11). Inhibitors of farnesyltransferase or geranylgeranyltransferase enzymes (FTIs and GGTIs, respectively) that attach isoprenoid lipids to proteins are being explored for treating malignancy(12-14), Hutchinson-Gilford progeria syndrome(15), malaria(16, 17) and other diseases. Identification of tissues and cell types targeted therapeutically by mevalonate pathway inhibitors that block protein prenylation remains a crucial but elusive goal. An important example is usually aminobisphosphonate-based adjuvant chemotherapy in breast cancer. Here, whether the antitumor activity of zoledronic acid or other aminobisphosphonates occurs by direct targeting of tumor cells or indirect targeting of osteoclasts or other host cell types remains unknown despite rigorous investigation(6, 7, 10, 18, 19). Such questions have persisted because surveying and quantifying drug efficacy and pharmacodynamics in tumors or numerous host organs, tissues and cell types in vivo has proved hard with biochemical methods used heretofore to assess prenylation inhibition(6, 7, 20). To eliminate this hurdle, we describe herein the development of a non-invasive, geneticallyencoded, bioluminescence-based imaging reporter that specifically and quantitatively detects direct targeting of living cells by prenylation inhibitors. We investigate the utility of this imaging reporter by introducing it into breast malignancy cells and determining whether unique classes of prenylation inhibitors can target tumor cells directly in mouse xenograft models of breast cancer. Materials and Methods Reagents MDA-MB-231 cells were obtained from Dr. Theresa Guise (Indiana University or college School of Medicine)(21). Drugs were obtained from the following sources: clodronate and GGTI-298 (Sigma-Aldrich), simvastatin (Calbiochem) and zoledronic acid (Novartis Pharma AG, Basel, Switzerland). Reporter Construction The VP16 transcriptional activation domain name from pVP16 (Clontech) was inserted downstream of the Gal4 DNA binding-domain coding region in pM3 (Clontech). The Gal4-VP16 coding region was inserted upstream of the GFP coding region in pEGFP-C1 (Clontech) to create a Gal4-VP16-GFP fusion. Oligonucleotides encoding the C-terminal 19 amino acids of Cdc42 with a functional (WT) or inactivated (CS) prenylation site were used to generate ITE plasmids encoding Gal4-VP16-GFP-Cdc42tail fusion proteins. The firefly luciferase (Fluc) coding region from pGL3 (Promega) was inserted into pcDNA6-V5/HisA (Invitrogen) with five copies of a consensus Gal4 DNA binding site. The Gal45-Fluc, ubiquitin C promoter/MCS/IRES/Renilla luciferase (from pRLTK (Promega)), the Gal4-VP16-GFP-Cdc42tail fragments, and ITE a PGKneo cassette from pPGKneo-I (Genbank accession #”type”:”entrez-nucleotide”,”attrs”:”text”:”AF335419″,”term_id”:”21955179″,”term_text”:”AF335419″AF335419) were inserted into plasmid FCIV for lentivirus packaging. HEK293T cells were co-transfected with the FCIV constructs, pVSVG, and D8.91 plasmids using Effectene (Qiagen) to generate lentivirus (pVSVG, D8.9 packing vector and the transfer vector FCIV were provided by J. Milbrandt; Washington University or college School of.For example, statins, which inhibit HMG-CoA reductase to treat hypercholesterolemia, are under investigation in malignancy and dementia(2-4). Introduction The mevalonate biosynthetic pathway operates in all human organs and cell types to ITE provide precursors for synthesizing steroids and isoprenoids that maintain cell membrane structure, function as endocrine hormones, produce heme A and ubiquinone for electron transport, or change proteins post-translationally with isoprenoid lipids (prenylation) or N-linked oligosaccharide chains(1). Mevalonate pathway inhibitors that blunt protein prenylation are being investigated intensively for treating cancer and other diseases. For example, statins, which inhibit HMG-CoA reductase to treat hypercholesterolemia, are under investigation in malignancy and dementia(2-4). Aminobisphosphonates, which inhibit farnesylpyrophosphate synthase (FPP synthase) in osteoclasts to reduce bone loss in osteoporosis and metastatic malignancy(5-8), are being studied preclinically and clinically as adjuvant chemotherapeutics that exert antitumor effects in breast cancer(9-11). Inhibitors of farnesyltransferase or geranylgeranyltransferase enzymes (FTIs and GGTIs, respectively) that attach isoprenoid lipids to proteins are being explored for treating cancer(12-14), Hutchinson-Gilford progeria syndrome(15), malaria(16, 17) and other diseases. Identification of tissues and cell types targeted therapeutically by mevalonate pathway inhibitors that block protein prenylation remains a crucial but elusive goal. An important example is aminobisphosphonate-based adjuvant chemotherapy in breast cancer. Here, whether the antitumor activity of zoledronic acid or other aminobisphosphonates occurs by direct targeting of tumor cells or indirect targeting of osteoclasts or other host cell types remains unknown despite intensive investigation(6, 7, 10, 18, 19). Such questions have persisted because surveying and quantifying drug efficacy and pharmacodynamics in tumors or various host organs, tissues and cell types in vivo has proved difficult with biochemical methods used heretofore to assess prenylation inhibition(6, 7, 20). To eliminate this hurdle, we describe herein the development of a non-invasive, geneticallyencoded, bioluminescence-based imaging reporter that specifically and quantitatively detects direct targeting of living cells by prenylation inhibitors. We investigate the utility of this imaging reporter by introducing it into breast cancer cells and determining whether distinct classes of prenylation inhibitors can target tumor cells directly in mouse xenograft models of breast BMP6 cancer. Materials and Methods Reagents MDA-MB-231 cells were obtained from Dr. Theresa Guise (Indiana University School of Medicine)(21). Drugs were obtained from the following sources: clodronate and GGTI-298 (Sigma-Aldrich), simvastatin (Calbiochem) and zoledronic acid (Novartis Pharma AG, Basel, Switzerland). Reporter Construction The VP16 transcriptional activation domain from pVP16 (Clontech) was inserted downstream of the Gal4 DNA binding-domain coding region in pM3 (Clontech). The Gal4-VP16 coding region was inserted upstream of the GFP coding region in pEGFP-C1 (Clontech) to create a Gal4-VP16-GFP fusion. Oligonucleotides encoding the C-terminal 19 amino acids of Cdc42 with a functional (WT) or inactivated (CS) prenylation site were used to generate plasmids encoding Gal4-VP16-GFP-Cdc42tail fusion proteins. The firefly luciferase (Fluc) coding region from pGL3 (Promega) was inserted into pcDNA6-V5/HisA (Invitrogen) with five copies of a consensus Gal4 DNA binding site. The Gal45-Fluc, ubiquitin C promoter/MCS/IRES/Renilla luciferase (from pRLTK (Promega)), the Gal4-VP16-GFP-Cdc42tail fragments, and a PGKneo cassette from pPGKneo-I (Genbank accession #”type”:”entrez-nucleotide”,”attrs”:”text”:”AF335419″,”term_id”:”21955179″,”term_text”:”AF335419″AF335419) were inserted into plasmid FCIV for lentivirus packaging. HEK293T cells were co-transfected with the FCIV constructs, pVSVG, and D8.91 plasmids using Effectene (Qiagen) to generate lentivirus (pVSVG, D8.9 packing vector and the transfer vector FCIV were provided by J. Milbrandt; Washington University School of Medicine). MDA-MB-231 cells were plated in 6-well dishes (2104 cells/well) with 8 mg/ml polybrene (Sigma) and infected with 4-500 ml of virus-containing medium. After 24h, cells were transferred into 10cm dishes. At 48h, 800 g/ml G418 (Sigma) was applied to select for stably transduced cells. Confocal Microscopy MDA-MB-231 cells were transfected (Lipofectamine 2000, Invitrogen) on coverslips.