Coexpression of a Multidrug-resistance Gene (MDR1) and Herpes Simplex Virus Thymidine Kinase Gene as Part of a Bicistronic Messenger RNA in a Retrovirus Vector Allows Selective Killing of MDR1-transduced Cells1

A new retroviral vector, pSXLC/pHa, was constructed to coexpress drug-selectable markers with a second gene of interest as a part of a bicistronic mRNA in a retroviral vector using an internal ribosome entry site (IRES) from encephalomyocarditis virus. This system was used to develop a new retroviral vector pHa-MDR-IRES-TK which expresses a single mRNA from which translation of the MDR1 gene is cap dependent and translation of the herpes simplex virus thymidine kinase gene is IRES dependent. The pHa-MDR-IRES-TK transfectants showed high levels of P-glycoprotein expression and multidrug resistance. More than 95% of the vincristine-resistant cells transfected or transduced with pHa-MDR-IRES-TK showed hypersensitivity to ganciclovir, which selects against cells expressing herpes simplex virus thymidine kinase. An amphotropic retrovirus titer of 7.8 x i0 /ml was obtained with this vector. This safety-modified vector should be useful for introducing the MDR1 gene into bone marrow cells to protect normal cells from the toxic effects of cancer chemotherapy because this vector allows the elimination of cancer cells that have been unintentionally transduced with the MDR1 vector. INTRODUCTION In cancer chemotherapy, there are two major problems to be overcome. One is the innate or acquired resistance of cancer Received 9/19/94; accepted 12/7/94. I This investigation has been aided by a grant from The Jane Coffin Childs Memorial Fund for Medical Research. 2 Recipient of a fellowship from the National Cancer Institute-Japanese Foundation for Cancer Research Research Training Program. Present address: Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan. 3 Fellow of The Jane Coffin Childs Memorial Fund for Medical Research. 4 To whom requests for reprints should be addressed, at Laboratory of Cell Biology, Building 37, Room 1B22, National Cancer Institute, National Institutes of Health, 37 Convent Drive MSC 4255, Bethesda, MD 20892-4255. cells to anticancer drugs, and the other is the toxicity of the chemotherapeutic drugs to certain normal tissues, such as bone marrow. The study of the mechanisms of drug resistance in cancer cells has led to the identification of some of the genes and gene products that confer drug resistance. In particular, cell lines showing resistance to multiple drugs such as Vinca alkabids, anthracyclines, epipodophyllotoxins, and actinomycin D have been studied intensively, and one gene responsible for this form of multidrug resistance, termed MDR1,5 has been identified and a full-length eDNA of the gene has been cloned and sequenced (1, 2). The MDRI gene encodes the plasma membrane P-glycoprotein with a molecular mass of 170 kDa. Pglycoprotein acts as an ATP-dependent efflux pump for various structurally unrelated natural product antitumor agents (reviewed in Ref. 3). The MDR1 eDNA has been shown to confer multidrug resistance when introduced into drug-sensitive cells (4, 5). The MDR1 gene is normally expressed on the biliary surfaces of hepatocytes, the brush border of the proximal tubules of kidney, the lumen of small and large intestine, and the capillary endothelial cells of the brain and testes (3). However, it is not widely expressed in bone marrow cells with the possible exception of some CD34-positive cells (6). Lack of protection by an endogenous transporter may be one of the reasons for the severe suppression of bone marrow cells by many chemotherapeutic drugs, which is a major dose-limiting factor in cancer chemotherapy. Retrovirus-mediated expression of the MDR1 cDNA has been shown to confer multidrug resistance in vivo when the MDR1-carrying vector is introduced into bone marrow cells of mice (7, 8). Studies using MDR1-transgenic mice suggest that the expression of the human MDR1 cDNA in bone marrow cells does not affect the normal function of the bone marrow cells (9, 10). Therefore, in principle, the MDR1 gene can be used to protect bone marrow cells from intensive chemotherapy. There still remain, however, some potential problems associated with MDR1 gene transfer into normal bone marrow cells. One problem is to optimize the efficiency of MDR1 gene transfer and expression. Using an MDR1 retrovirus, relatively efficient MDR1 gene transfer is possible, but development of higher titer retroviruses and an optimized protocol for selection of the transduced cells in vivo are still required. Another prob5 The abbreviations used are: MDR, multidrug-resistance gene; HSVTK, herpes simplex virus thymidine kinase; IRES, internal ribosome entry site; TK, thymidine kinase; LTR, long terminal repeat; IC5 , concentration of drug that inhibits cell growth by 50%; FACS, fluorescence-activated cell sorting; HAT, hypoxanthine-aminopterin-thymidine; MCS, multicloning site. Research. on November 11, 2017. © 1995 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 448 Retroviral Coexpression of MDR1 and HSV-TK lem is that it is possible to transduce other cells contained within bone marrow preparations in which the expression of the transduced gene might cause undesirable side effects. In transducing bone marrow cells of cancer patients with the MDR1 retrovirus, if the bone marrow contains contaminating cancer cells and the cancer cells receive the MDR1 retrovirus, then multidrug-resistant cancer cells will result. In such cases, treatment protocols involving drugs related to multidrug resistance will be ineffective. To eliminate such unintentionally transduced cells, introduction of a negative drug-selectable marker (a ‘ ‘suicide’ ‘ gene) that confers hypersensitivity to a certain drug would be valuable. The HSV-TK gene acts as a suicide gene both in vitro and in vivo. Cells that express HSV-TK are hypersensitive to the nucleoside analogue ganciclovir because HSV-TK can phosphorylate ganciclovir more efficiently than the endogenous TK of mammalian cells. Ganciclovir treatment was reported to be effective in vivo against rat cerebral gliomas transduced with the retrovirus carrying HSV-TK (ii, 12). Multidrug-resistant cells do not show cross-resistance to ganciclovir. Therefore, the HSV-TK gene would appear to be a suitable gene to coexpress with the MDR1 gene to eliminate cancer cells transduced with the MDR1 vector. HSV-TK can also confer HAT resistance in vitro when it is introduced into TK-deficient cells such as Ltk There are three possible strategies for coexpression of the MDR1 gene with the HSV-TK gene. The first strategy is to utilize two independent promoters (e.g., retroviral LTR and another promoter). However, this does not guarantee the expression of both genes. Most transduced cells express only one gene because expression of one gene may suppress the activity of the second promoter (13). The second strategy is to engineer a chimeric bifunctional protein between P-glycoprotein and HSVTK. This approach has been shown to be successful when a second gene product, such as adenosine deaminase, is connected to the carboxyl terminus of P-glycoprotein (14, 15). This strategy guarantees the coexpression of the two gene products; however, the activity of chimeric P-glycoprotein as a drug transporter is less than the intact P-glycoprotein and some gene products may not function when tethered to the inside of the plasma membrane. The third strategy is to use an IRES isolated from a picornavirus such as encephalomyocarditis virus (1618). In this construct, a single mRNA is transcribed under the control of an upstream promoter, and two gene products are translated independently from the mRNA. The first open reading frame is translated in a cap-dependent fashion, and the second is translated under control of the IRES. We have developed a new retroviral vector system, pSXLC/pHa, in which the drug-selectable genes such as the MDR1 gene or the HSV-TK gene is translated under control of the IRES (19). To express the MDR1 gene and the HSV-TK gene together, we constructed a new MDR1-retrovirus vector pHa-MDR-IRES-TK that allows simultaneous translation of the cap-dependent MDRJ gene and the IRES-dependent HSV-TK gene. MATERIALS AND METHODS Cell Culture and Drug Sensitivity Assay. The edotropic retrovirus packaging cell line I’-cre, the amphotropic retrovirus packaging cell line P-crip (20), and the mouse fibroI LTRF_1MDRJ_H LTRI pHaMDR I LTR IRES TK xi : _ 1 LTR pHa-MCS-IRES-TK I LTRHIMD4IRESI TK1__-1L1 RI pHa-MDR-IRES-TK Fig. I Structure of pHaMDR, pHa-MCS-IRES-TK, and pHa-MDRIRES-TK retrovirus. Drawing is not to scale. LTR, LTR of Harvey murine sarcoma virus; MDR, human multidrug resistance gene MDR1; TK, herpes simplex virus thymidine kinase gene; S, Sacli; Xh, XhoI; MCS, SacII-BamHI-BglII-SacI-XbaI-SalI; N, NcoI; Xb, XbaI. blast cell line NIH3T3 were cultured in DMEM supplemented with 10% calf serum. The murine TK-deficient cell line Ltk and the amphotropic retrovirus packaging cell line PA317 (21) were grown in DMEM supplemented with 10% fetal bovine serum. The sensitivities of the cultured cell lines to drugs were evaluated by the inhibition of cell growth after incubation at 37#{176}C for 6 days in the presence of various concentrations of drugs as described previously (22). Cell numbers were determined in a Coulter counter and the IC50 was calculated. Construction of Vectors. The construction of the pSXLC/pHa retrovirus system was described previously (19). The plasmid pSXLC-TK has the entire open reading frame of HSV-TK DNA downstream from the IRES sequence and 6 unique sites (SaclI, BamHI, BglII, Sad, XbaI, and SalI) for the cloning of another gene upstream from the IRES. To insert the MDR1 eDNA into pSXLC-TK, we made pSXbaMDR, which contains MDR1 eDNA with 5’-SacII and 3’-XbaI sites in a pGEM2-derived vector, then subcloned the SacII-XbaI-digested MDR1 eDNA between the SaclI and XbaI sites of pSXLC-TK (pSXLC-MDR-TK). The pSXLC-MDR-TK insert was isolated after SacII-XhoI digestion and transferred into the pHa retroviral vector (pHa-MDR-IRES-TK). pHaMDR, which carries the wild-type MDR1 eDNA in the pHa retrovirus vector (5, 23), was used as a control MDR1 vector. pHa-MCS-IRES-TK, which carries the insert of pSXLC-TK, was also used as a control (19). Structures of retrovirus constructs used in this study are summarized in Fig. 1. DNA Transfection. Transfection was carried out using the calcium phosphate coprecipitation method (24). Recipient cells were plated at 5 x i0 cells/100-mm dish on day 1 and transfected with 20 jig of the expression plasmid DNA on day 2. Cells were exposed to the DNA precipitate until day 3 when the medium was aspirated and fresh medium was added. On day 4, the cells were split at 1:10 or 1:100. The cells were selected either in vincristine (25 ng/ml for ‘I’-cre, ‘P-drip, and NIH3T3; 30 ng/ml for PA317 and 35 ng/ml for Ltk) or HAT medium (100 p.M hypoxanthine, 0.4 p.M aminopterin, and 16 p.M thymidine) on day 5, if necessary. Retrovirus Transduction. To examine the production of retrovirus, retrovirus-producing cells were plated on day 1 at Research. on November 11, 2017. © 1995 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Clinical Cancer Research 449 2 x 106 cells/100-mm dish. On day 2, the medium of the packaging cell culture was changed, and the recipient cells were plated at 3 x iO cells/100-mm dish in medium containing 2 p.g/ml polybrene (Aldrich, Milwaukee, WI). On day 3, the retrovirus-containing supernatant was collected, passed through a 0.45-jim pore filter to remove cells and debris, and added to each dish of recipient cells. On day 5, the medium was removed, and fresh medium containing drugs was added at appropriate concentrations as needed. To estimate the retrovirus titer, the medium was removed and the colonies were stained with 0.5% methylene blue dissolved in 50% methanol on days 11-13. Protein Expression Analysis. To examine the expression of human P-glycoprotein on the cell surface of mouse transfectants, FACS analysis was used in which cells were reacted with a human P-glycoprotein-specific mAb MRK16 (25, 26). Cells (106) harvested after trypsinization were washed and incubated with MRK16 (5 p.g/106 cells), washed twice, and incubated with fluorescein-conjugated goat anti-mouse IgG (1:10 diluted; Jaxon Immunoresearch Lab., West Grove, PA). The cells were washed twice and the fluorescence staining level was analyzed using a FACSort (Becton Dickinson FACS System, San Jose, CA). Nucleic Acid Isolation and Analysis. Total RNA was isolated from cultured cells by acid-guanidinium-thiocyanatephenol-chloroform extraction (Stratagene, Inc., La Jolla, CA; Ref. 27). Cells were first homogenized by passage through a 20-gauge needle after removal from the dish by direct addition ofthe guanidinium thiocyanate. For Northern blot hybridization, RNA was resolved on a 1% formaldehyde-agarose gel and subsequently transferred to nitrocellulose (BA83; Schleicher & Schuell) (28). The blot was probed with a random-primed 0.8kilobase MDR1 probe obtained from an EcoRI-HindIII digestion of MDR1 cDNA (29). The blot was subsequently stripped and reprobed with a random-primed 1.2-kilobase NcoI-XhoI fragment from pSXLC-TK containing the entire open reading frame for HSV-TK. Hybridization and washing conditions were as described previously (30). RESULTS We have been using the pHaMDR retroviral vector which uses the promoter of the Harvey murine sarcoma virus LTR to drive expression of the MDR1 gene in mammalian cells (5, 31). In a previous study we reported the generation of a retroviral vector system pSXLC/pHa that uses the IRES sequence to coexpress drug-selectable genes with the gene of interest (19). To coexpress the HSV-TK gene as a suicide gene in the pHa vector, we made pSXLC-TK with the entire open reading frame of the HSV-TK gene downstream from the IRES and six unique sites (Sad!, BamHI, BglII, Sad, XbaI, and Sail) for the cloning of another gene (MDR1) upstream from the IRES. This IRES-TK construct was shown to be effective when the insert of pSXLC was transferred into the pHa vector (pHa-MCS-IRESMDR) and introduced to Ltk cells (19). In the current study, we inserted the MDR1 cDNA into pSXLC-TK upstream from the IRES (pSXLC-MDR-TK) and transferred the whole insert into the pHa vector. The resulting construct was termed pHaMDR-IRES-TK (Fig. 1). Table I Transfection efficiency of pHa-MDR-IRES-TK and pHaMDR Cell Vector Transfection efficiency”