Targeted expression of human folylpolyglutamate synthase for selective enhancement of methotrexate chemotherapy in osteosarcoma cells

Abstract

The antifolate methotrexate (MTX) is an important chemotherapeutic agent for treatment of osteosarcoma. This drug is converted intracellularly into polyglutamate derivates by the enzyme folylpolyglutamate synthase (FPGS). MTX polyglutamates show an enhanced and prolonged cytotoxicity in comparison to the monoglutamate. In the present study, we proved the hypothesis that transfer of the human fpgs gene into osteosarcoma cells may augment their MTX sensitivity. For this purpose, we employed the human osteocalcin (OC) promoter, which had shown marked osteosarcoma specificity in promoter studies using different luciferase assays in osteosarcoma and non-osteosarcoma cell lines. A recombinant lentiviral vector was generated with the OC promoter driving the expression of fpgs and the gene for enhanced green fluorescent protein (egfp), which was linked to fpgs by an internal ribosomal entry site (IRES). As the vector backbone contained only a self-inactivating viral LTR promoter, any interference of the OC promoter by unspecific promoter elements was excluded. We tested the expression of FPGS and enhanced green fluorescent protein (EGFP) after lentiviral transduction in various osteosarcoma cell lines (human MG-63 cells and TM 791 cells; rat osteosarcoma (ROS) 17/2.8 cells) and non-osteogenic tumor cell lines (293T human embryonic kidney cells, HeLa human cervix carcinoma cells). EGFP expression and MTX sensitivity were assessed in comparison with non-transduced controls. Whereas the OC promoter failed to enhance MTX sensitivity via FPGS expression in non-osteogenic tumor cell lines, the OC promoter mediated a markedly increased MTX cytotoxicity in all osteosarcoma cell lines after lentiviral transduction. The present chemotherapy-enhancing gene therapy system may have great potential to overcome in future MTX resistance in human osteosarcomas.

Introduction

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Surgical resection and chemotherapy are mainstays of today’s therapy. More than 40% of patients diagnosed with osteosarcoma will fail this conventional first line therapy and develop progressive or recurrent disease with an overall poor prognosis.1, 2

One approach to improve this situation may be to optimize therapeutic efficiency of the current chemotherapy protocols. The antifolate methotrexate (MTX) represents the central chemotherapeutic agent in most osteosarcoma treatment protocols worldwide. It is commonly used at very high doses because lower MTX doses were shown to be associated with a worse outcome.3 This clinical observation might indicate an intrinsic resistance mechanism against antifolates in osteosarcoma. Looking for such intrinsic resistance mechanisms, it was reported that decreased expression of the reduced folate carrier (solute carrier family 19A1, SLC19A1) resulting in decreased uptake of MTX was found especially in those osteosarcomas with a poor response to chemotherapy.4, 5

In acute lymphoblastic leukemias where MTX also represents an essential chemotherapeutic agent, reduced activity of the enzyme folylpolyglutamate synthase (FPGS) was mainly associated with MTX resistance.6, 7 FPGS is responsible for enhanced therapeutic efficiency of MTX by converting intracellular MTX monoglutamates into polyglutamates. Polyglutamination increases the intracellular amount of MTX because ionized MTX polyglutamates cannot effectively cross cell membranes. Furthermore, affinity and inhibition of MTX polyglutamates for key enzymes of folate metabolism, for example, dihydrofolate reductase, are also enhanced.8

In osteosarcomas, the role of FPGS for MTX resistance and clinical outcome has not been studied yet. Thus, it is not known if transfer of FPGS into osteosarcoma cells may augment therapeutic efficiency of MTX, as it was shown for glioma and breast cancer cells.9, 10 In the present study, human osteosarcoma cell lines MG-63 and TM 791 as well as rat osteosarcoma cell line ROS 17/2.8 were used as in vitro models for a chemotherapeutic drug-modulating gene therapy employing FPGS and MTX. The osteocalcin (OC; bone gamma-carboxyglutamate protein) promoter was employed for transcriptional targeting of FPGS expression mainly to osteosarcoma cells. This promoter had been previously shown to be highly active in osteosarcoma cells.11 A lentiviral vector system was used to ensure sufficient levels of FPGS transduction efficiencies in various in vitro models.

The present model of targeted FPGS/MTX chemogene therapy in osteosarcoma cells may indicate the great therapeutic potential of pharmacogenetic findings in tumors and other diseases, which may be more widely used in the future to selectively modulate therapeutic effects of conventional drug therapies in target cells.

Materials and methods

Cell lines

The human osteosarcoma cell lines MG-6312 and TM 791,13 the rat osteosarcoma cell line ROS 17/2.8,14 the human cervix carcinoma cell line HeLa15 and the human embryonic kidney cell line 293T16 were cultured in Dulbecco’s modified Eagle’s Medium (Life Technologies, Gaithersberg, MD, USA) supplemented with penicillin (100 units ml−1), streptomycin (100 μg ml−1), L-Glutamine (Sigma Chemical, Taufkirchen, Germany), and 10% fetal bovine serum (Sigma).

Expression vectors

A 604-bp human OC cDNA fragment including the 5′ flanking region with the OC promoter was amplified from DNA from cell line MG-63 by polymerase chain reaction using primers 5′-IndexTermAAG CTT GCT GAC CGT CGA GCT GCA C-3′ and 5′-IndexTermGGC TCT CCT GGT GTC TCG-3′ and subsequently inserted into the promotor-less luciferase gene expression vector pGL3-BASIC (Promega, Mannheim, Germany). The resulting new expression vector was designated as pGL3-OC (Figure 1a).

Figure 1
figure1

Schematic representation of the used constructs. (a) Schematic representation of luciferase constructs. The osteocalcin (OC) promoter (POC) was isolated from cell line MG-63 by polymerase chain reaction and cloned into the luciferase-expressing vector PGL3 by the use of flanking EcoRI sites. The vector contains an ampicillin resistance gene (Amp). In the pGL3-CONTROL vector, luciferase is controlled by the SV40 Promoter (PSV40). This plasmid was used as the positive control. The pGL3-BASIC vector does not contain any promoter elements and was used as negative control. (b) Schematic representation of pLNT-CMV-FPGS-EGFP, pLNT-OC-FPGS-EGFP and pcDNA HIV CS-CGW. pLNT-CMV-FPGS-EGFP and pLNT-OC-FPGS-EGFP contain the transcriptional unit FPGS-IRES-EGFP. The only difference between these two constructs is the promoter, the (cytomegalovirus) CMV (PCMV) or the OC promoter (POC). pcDNA HIV CS-CGW17 served as a control vector.

An expression cassette containing a FPGS fragment (kindly provided by Dr B Shane, University of California, Berkeley, CA, USA) under the control of the human OC promoter was cloned into plasmid pIRES2-EGFP (Clontech, Heidelberg, Germany). The resulting new expression vector was designated pOC-FPGS-EGFP. A 3805-bp fragment containing the gene expression cassette FPGS-IRES-EGFP was cloned and finally inserted into expression plasmid pEGFP-N1 (Clontech). The resulting new expression plasmid was designated pCMV-FPGS-EGFP. Using the Gateway-Cloning technology (Invitrogen, Karlsruhe, Germany) both FPGS-EGFP co-expression cassettes either under control of the CMV promoter (pCMV-FPGS-EGFP) or of the OC promoter (pOC-FPGS-EGFP) were transferred into a modified lentiviral vector pLNT-MCS (kindly provided by Prof. Dr A Thrasher, Institute of Child Health, London, UK), containing the Gateway attL recombination sites, respectively. The resulting lentiviral vectors were designated pLNT-CMV-FPGS-EGFP and pLNT-OC-FPGS-EGFP, respectively (Figure 1b). The lentiviral vector pcDNA HIV CS-CGW17 (Figure 1b) containing the egfp gene under control of the CMV promoter was used as a control vector.

Luciferase assays

Luciferase reporter gene expression was analyzed in osteosarcoma and control cells using the Dual-Luciferase- and Single-Luciferase-Reporter Assay Systems (Promega, Mannheim, Germany). All transfections with the various pGL3 constructs (pGL3-BASIC, pGL3-CONTROL, pGL3-OC) were carried out using FuGENE 6 transfection reagent (Roche, Mannheim, Germany). Luciferase activities were determined 48 h after transfection using a Microlumat LB96P luminometer (Berthold, Bad Wildbad, Germany). In dual-luciferase assays, activity of firefly luciferase was normalized to the activity of control Renilla luciferase for each cell line to rule out possible bias due to different transfection efficiencies. For single-luciferase assays, firefly luciferase activities were normalized to the respective protein concentration of each sample (BioRad protein assay, BioRad Laboratories, Munich, Germany).

Lentivirus generation and transduction

Lentivirus containing supernatants of pLNT-CMV-FPGS-EGFP, pLNT-OC-FPGS-EGFP, and pcDNA HIV CS-CGW were generated by FuGENE 6-mediated triple-transfection of 293T cells with the packaging plasmid pCMVΔR8.91, the envelope-plasmid pVSV-G and the gene-transfer-plasmids pLNT-CMV-FPGS-EGFP, pLNT-OC-FPGS-EGFP or pcDNA HIV CS-CGW, respectively. Seventy-two hours after transfection, virus particles were harvested, subsequently concentrated by centrifugation, and stored at−80 °C following standard procedures.18 Viral stocks were tested on cell lines HeLa and ROS 17/2.8 by flow cytometric evaluation of EGFP-positive cells 72 h after infection.

Lentiviral infection of mammalian cells

All cell lines (ROS 17/2.8, TM 791, MG-63, HeLa, 293T) were plated at 2.5 × 104 cells per well in 12-well plates. After 12 h, medium was removed and 500 μl of lentiviral supernatants (pLNT-CMV-FPGS-EGFP, pLNT-OC-FPGS-EGFP, and pcDNA HIV CS-CGW) with 1 × 106 infectious particles was added to the cells. Twenty-four hours after infection, 1 ml medium was added. The infection rate was determined 72 h after infection by counting EGFP-positive cells by use of a FACS-Calibur (Becton-Dickinson, Heidelberg, Germany).

Cell survival studies

Cells were plated in triplicates at 1 × 105 cells per well in 6-well plates. Cells were allowed to adhere for 16 h, after which the medium was replaced with medium containing MTX at a concentration of 0, 0.1, 0.5, 1, 5, or 10 nmol ml−1. After 4 h of drug exposure, plates were washed three times with phosphate-buffered saline. Drug-free medium was then added, and 72 h later, cells were trypsinized and counted using a Neubauer chamber.

Results

Osteosarcoma-selective promoter activity in transiently transfected cells

In order to investigate the feasibility of the OC promoter system for osteosarcoma-selective gene expression, we generated constructs containing the reporter gene luciferase under the control of the human OC promoter (Figure 1a). We studied reporter gene expression in transient transfection experiments using human and rat osteosarcoma cell lines as well as a cervix carcinoma cell line as negative control. Constructs that contained the constitutive SV40 promoter or a promoter-less construct were used as additional controls (Figure 1a). The luciferase reporter gene assays were performed both as single and dual-luciferase assays. In the dual-luciferase assay, activity of the constitutively expressed Renilla luciferase was used as internal control to normalize the activity of the specifically expressed firefly luciferase to minimize the possible bias as a result of variations in transfection efficiencies. The dual-luciferase assay results are summarized in Figure 2. The additional single luciferase assays revealed similar results, thus indicating that the activities obtained with the dual assay were not altered by promoter interferences (data not shown). To increase OC promoter activity, we investigated luciferase expression after inducing the promoter with vitamin D. The luciferase reporter gene assays revealed an osteosarcoma-selective expression by the OC promoter. The vitamin D treatment increased the luciferase expression by the OC promoter in both osteosarcoma cell lines. In contrast, the OC promoter allowed no luciferase expression in the non-osteogenic HeLa cell line (Figure 2).

Figure 2
figure2

Cell-specific osteocalcin (OC) promoter activity. Indicated cell lines were transiently transfected with different pGL3 constructs and treated with or without vitamin D3 (vitD3). Forty-eight hours after transfection, luciferase assays were performed. For calculation of relative luciferase activity, luciferase activity of cells transfected with the control plasmid pGL3-CONTROL was set as 100%. The data represent means from 2–5 independent dual-luciferase assays.

Cloning and production of lentiviral vectors expressing FPGS-IRES-EGFP

On the basis of the encouraging results of the transient transfection experiments, the OC promoter was investigated further in the context of an osteosarcoma-selective chemogene therapy approach using the FPGS/MTX system. We used a three-plasmid expression system to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells with the defective packaging plasmid pCMVΔR8.91, a plasmid coding for the envelope protein of the vesicular stomatitis virus (pVSV-G), and a vector construct (pLNT) coding for the expressing cassettes. The packaging plasmid pCMVΔR8.91 lack functional vif, vpr, and vpu proteins and a large portion of the envelope coding region as well as the 5′ and 3′ long terminal repeats, the nef function, and the presumed packaging signal. The resulting infectious lentiviral particles were tested on the cell lines HeLa and the ROS 17/2.8 by counting EGFP-positive cells 72 h after infection. To minimize the possible bias as a result of variations of the cell-surface receptors and various promoter activities, all lentiviral supernatants were measured by a p24-enzyme-linked immunosorbent assay (kindly performed by O Adams, Institute for Virology, Heinrich Heine University, Duesseldorf). As expected, there was no measurable EGFP expression after the infection of 293T cells with the vector pLNT-OC-FPGS-EGFP. Therefore, it was not possible to determine the titer of the lentiviral OC promoter plasmid by infection of 293T cells.

However it was possible to determine the titer of vector pLNT-OC-FPGS-EGFP by infecting ROS 17/2.8 cells. Titers of 5 × 106 infectious particles per ml were detected. After infection with the non-specific pLNT-CMV-FPGS-EGFP construct, titers of 5 × 106 infectious particles per ml were measured in 293T cells as well as in ROS 17/2.8 cells. The results of the p24-enzyme-linked immunosorbent assay, which are independent of variations of the cell-surface receptors and various promoter activities, were all in the same range (4400–5800 ng ml−1), indicating that there were no large variations in the concentration of the different lentiviral supernatants (Table 1).

Table 1 Comparison of supernatants used for transduction of cell lines

Generation of stably transduced osteosarcoma and non-osteogenic cell lines by lentiviral transduction

To investigate the specificity of OC promoter-mediated chemogene therapy, we established an in vitro model by stable lentiviral transduction of osteosarcoma and non-osteogenic cell lines. The human osteosarcoma cell lines ROS 17/2.8, MG-63, and TM 791 as well as the non-osteogenic cell lines HeLa and 293T were infected with lentiviral supernatants pLNT-OC-FPGS-EGFP, pLNT-CMV-FPGS-EGFP, and pcDNA HIV CS-CGW. After infection, EGFP expression of the resulting cell populations was determined by flow cytometry. Seventy-two hours after infection with the lentiviral supernatants, both CMV promoter constructs (pcDNA HIV CS-CGW and pLNT-CMV-FPGS-EGFP) showed in all cell lines a high EGFP expression (between 70 and 95% EGFP-positive cells). In contrast, the OC promoter construct (pLNT-OC-FPGS-EGFP) induced EGFP expression only in the osteosarcoma cells (between 60 and 70% EGFP-positive cells). In the ROS 17/2.8 cells, the OC promoter construct reached same levels of EGFP expression as the CMV promoter construct (pLNT-CMV-FPGS-EGFP). In MG-63 and TM 791 cells, the EGFP expression was slightly weaker, only 60–65% of the cells were EGFP-positive. However, there was a clear difference to the OC-negative cell lines. Here, the percentage of the EGFP-positive cells after infection with pLNT-OC-FPGS-EGFP was only 5% (Figures 3 and 4).

Figure 3
figure3

Enhanced green fluorescent protein (EGFP) expression after lentiviral transduction of different cell lines. Indicated cell lines were transduced with the different lentiviral constructs. Expression of EGFP was assessed by flow cytometry.

Figure 4
figure4

Enhanced green fluorescent protein (EGFP) expression after lentiviral transduction of different cell lines. Indicated cell lines were transduced with the different lentiviral constructs or left untreated (native). Presented are representative flow cytometry results for an osteocalcin-negative cell line (HeLa) and an osteocalcin-positive cell line (ROS 17/2.8). Cytomegalovirus (CMV)=cells transduced with control vector pCDN HIV CS-CGW; CMV-FPGS=cells transduced with vector pLNT-CMV-FPGS-EGFP; OC-FPGS=cells transduced with vector pOC-CMV-FPGS-EGFP.

OC promoter mediates enhanced MTX sensitivity in osteosarcoma cells

Finally, we investigated the OC promoter-mediated cytotoxicity of MTX treatment in various stably transduced osteosarcoma cell lines (TM 791, MG-63, and ROS 17/2.8) and non-osteogenic cell lines (HeLa and 293T). We used the cells that were infected with pLNT-CMV-FPGS-EGFP supernatant as positive controls. This infection should induce a higher MTX toxicity in all cell lines. In addition, the cells that were infected with pcDNA HIV CS-CGW supernatant were used as negative controls because this plasmid was constructed without the FPGS as suicide gene, so that this infection should induce no higher MTX toxicity in all cell lines. Non-infected (native) cells were used as additional negative controls to reveal the toxicity of the MTX treatment without any genetic manipulations. The MTX sensitivity of the pcDNA HIV CS-CGW-infected cells did not differ significantly from the non-infected (native) cells, indicating that a cytotoxic effect triggered by the EGFP expression or the lentiviral infection by itself could be excluded. The pLNT-CMV-FPGS-EGFP-infected cells showed in all examined cell lines a significant increase of the MTX sensitivity. The pLNT-OC-FPGS-EGFP infection induced only in the osteosarcoma cell lines a significant increase of the MTX sensitivity. There was no increase in the non-osteogenic cell lines (Figure 5).

Figure 5
figure5

Methotrexate (MTX) sensitivity of cell lines after lentiviral transfer of folylpolyglutamate synthase (FPGS). Untreated (native) cells as well as cells that were infected with the three different lentiviral constructs were examined. All cells were treated with MTX over a period of 4 h. Seventy-two hours after the MTX treatment, the number of survived cells was determined.

Discussion

The aim of the present study was the development of a tumor-selective chemogene therapy approach for osteosarcoma. Enhancement of conventional MTX chemotherapy should be achieved through transfer of the fpgs gene, which had been shown to enhance MTX and other anti-folate-induced cytotoxicity in glioma and breast cancer cells.9, 10 Selectivity of this approach for osteosarcomas was intended by transcriptional targeting via the OC promotor, which is highly active in bone and dentin-forming cells in human tissues.

The OC promotor had been tested before in various models of selective osteosarcoma suicide gene therapy.11, 19, 20 Infection of osteosarcoma cells with an adenovirus containing Herpes simplex virus type 1 thymidine kinase under the control of the OC promoter resulted in an increased sensitivity for treatment with acyclovir.11 The effects were not seen in non-osteogenic cell lines, which were infected with the same adenovirus. Similarly, animals with metastatic lung disease of an osteosarcoma cell line (ROS 17/2.8) were successfully treated by using an adenovirus mediated gene therapy in which Herpes simplex virus type 1 thymidine kinase was also driven by the OC promoter.20 These preclinical studies served as a basis for the design of first clinical studies. Patients with chemotherapy-resistant lung metastases of an osteosarcoma were treated with an adenovirus. The OC promoter was used to regulate the expression of the adenoviral E1A protein leading to selective replication of the virus in tumor cells and subsequent tumor cell lysis.21 In two further clinical studies, an OC promoter-based cytotoxic gene therapy for patients with OC-positive bone metastases of prostate carcinoma was tested.22, 23 The promoter fragments used in the mentioned studies had been characterized extensively.24, 25, 26 The best promoter activity was observed with a 604-bp fragment containing the region from bp −531 to +56 relative to the transcription start of the OC gene.26 Thus, we used this fragment in our study.

As a first step, the specificity of the cloned OC promoter fragment was investigated by using the promotor fragments for luciferase reporter gene assays in osteosarcoma and non-osteogenic tumor cell lines. A clear osteosarcoma-specific luciferase activity could be shown. Furthermore, the OC promoter also contains a vitamin D-responsive element at the position −513 to −493 relative to the transcription start site of the OC gene,24 and it was shown that 1,25-dihydroxy vitamin D3 was able to upregulate OC promoter activity.24, 25, 27 In our own luciferase reporter gene assays, we confirmed enhancement of OC promoter activity by 1,25-dihydroxy vitamin D3 in OC-positive osteosarcoma cells, but not in non-osteogenic tumor cells.

As expected, the efficiency of transient transfection was low, especially for human MG-63 osteosarcoma cells. As high transfection rates and a stable transgene expression over several days are absolutely necessary for cytotoxicity assays, we chose a lentiviral vector system for further cell culture experiments. Retroviral vectors were shown to be less effective in similar promotor-specific cytotoxicity studies employing neuroblastoma cells.28 Furthermore, clinical suicide gene studies had also failed probably due to a low overall transduction efficiency by the used retroviral vectors.29 For the present study, we tried to achieve similar transduction efficiencies in all examined cell lines. To monitor the gene transfer, we visualized transgene expression by using the reporter EGFP.30 EGFP was linked with the chemogene fpgs by using an IRES.31 By this IRES, both EGFP and FPGS are expressed under the control of the same promoter. In contrast to a fusion gene, both genes are translated as two different proteins. However, the disadvantage of an IRES is the problem that the expression of the gene that is located downstream of the IRES can be reduced in comparison to the expression of the upstream gene.32 This reduced expression can lead to an underestimation of the transduction efficacy. As the use of a lentiviral vector system generally provides a strong gene expression, we did not see a problem in using the IRES. In fact, in all experiments we saw a distinct EGFP expression, thus, allowing us to correlate the extent of EGFP expression with gene transfer efficiency as well as with lentiviral titers in the various supernatants of lentiviral producer cells. Lentiviral infection of the different cell lines with similar virus titers resulted in high and stable transduction rates in all cell lines, thus allowing reliable cytotoxicity studies.

After lentiviral infection with the unspecific pCMV/fpgs constructs, MTX sensitivity was increased in all cell lines independently of their OC status. However, transcriptional targeting of fpgs expression by the OC promoter resulted in increased MTX-mediated cytotoxicity only in OC-positive osteosarcoma cells. In all FPGS-expressing tumor cells, MTX-mediated cytotoxicity appeared already at a MTX concentration of 0.5 nmol ml−1 (0.5 μmol l−1), which is a usually achievable serum level in MTX-treated patients. According to standardized treatment protocols, osteosarcoma patients are usually treated with high-dose-MTX, thereby achieving maximum MTX serum levels of 1000 μmol l−1 and higher. High MTX serum levels often result in significant toxicity to the skin, mucosa, kidney, liver, and bone marrow. To avoid increased toxicity to normal tissue and organs, MTX should be cleared from the patient’s circulation within a period of 4–7 days as shown by decrease of MTX serum levels to below 0.05 μmol l−1. The results of the present study suggest that osteosarcoma-selective fpgs chemogene therapy could markedly increase the anti-tumor efficiency of MTX in transduced osteosarcoma cells. Thus, even lower MTX doses can induce sufficient anti-osteosarcoma cytotoxicity with the potential of overall reduced chemotherapy-related side effects. Future studies are required in order to analyze if our lentiviral-mediated chemogene therapy approach also confers osteosarcoma-selective cytotoxicity in vivo.

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Acknowledgements

This work was supported by the Elterninitiative Kinderkrebsklinik Düsseldorf e.V. We thank O Adams, Düsseldorf, for performing p24-enzyme-linked immunosorbent assay.

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Correspondence to C M Kramm.

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Bienemann, K., Staege, M., Howe, S. et al. Targeted expression of human folylpolyglutamate synthase for selective enhancement of methotrexate chemotherapy in osteosarcoma cells. Cancer Gene Ther 20, 514–520 (2013). https://doi.org/10.1038/cgt.2013.48

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Keywords

  • osteosarcoma
  • gene therapy
  • FPGS
  • osteocalcin
  • methotrexate

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