Abstract
Human cells exposed to antifolates show a rapid increase in the levels of the enzyme dihydrofolate reductase (DHFR). We hypothesized that this adaptive response mechanism can be used to elevate cellular levels of proteins fused to DHFR. In this study, mouse cells transfected to express a green fluorescent protein-DHFR fusion protein and subsequently exposed to the antifolate trimetrexate (TMTX) showed a specific and time-dependent increase in cellular levels of the fusion protein. Next, human HCT-8 and HCT-116 colon cancer cells retrovirally transduced to express a DHFR-herpes simplex virus 1 thymidine kinase (HSV1 TK) fusion protein and treated with the DHFR inhibitor TMTX exhibited increased levels of the DHFR-HSV1 TK fusion protein and an increase in ganciclovir sensitivity by 250-fold. The level of fusion protein in antifolate-treated human tumor cells was increased in response to a 24-h exposure of methotrexate, trimetrexate, as well as dihydrofolate. This effect depended on the antifolate concentration and was independent of the fusion-protein mRNA levels, consistent with this increase occurring at a translational level. In a xenograft model, nude rats bearing DHFR-HSV1 TK-transduced HCT-8 tumors and treated with TMTX showed, after 24 h, a 2- to 4-fold increase of fusion-protein levels in tumor tissue from treated animals compared with controls, as determined by Western blotting. The fusion-protein increase was imaged with positron-emission tomography, where a substantially enhanced signal of the transduced tumor was detected in animals after antifolate administration. Drug-mediated elevation of cellular DHFR-fused proteins is a very useful method to modulate gene expression in vivo for imaging as well as therapeutic purposes.
Dihydrofolate reductase (DHFR; E.C. 1.5.1.3.) catalyzes the NADPH-dependent formation of 5,6,7,8-tetrahydrofolate (THF) from 7,8-dihydrofolate (DHF). The most significant consequence of DHFR inhibition by methotrexate (MTX) or trimetrexate (TMTX) is a decrease of thymidylate biosynthesis by means of depletion of the N5,N10-methylene-THF pool resulting in DNA synthesis inhibition and cell death. MTX is used to treat acute lymphoblastic leukemia, lymphoma, gastrointestinal cancers, and breast cancer. However, antifolate resistance remains an obstacle to successful cancer treatment. Early studies showed that treatment of leukemia patients with MTX increased DHFR activity in blast cells by 6-fold (1). Subsequently, Hillcoat et al. (2) observed a 12-fold increase in DHFR activity of cultured lymphoblasts after a 24-h exposure to MTX. Importantly, the increase of activity after 24–48 h was not affected by the addition of actinomycin D, but was inhibited by the addition of cycloheximide. Later studies (3, 4) showed no alteration of DHFR enzyme in human cells either treated with MTX in vitro or isolated from patients treated with MTX. Furthermore, mRNA levels were not increased after MTX treatment, and the half-life of DHFR in human cells was not affected by the presence or absence of MTX (4). Recent mechanistic studies from our laboratory and others indicate that DHFR binds to DHFR mRNA in the coding region, and that inhibition of DHFR by MTX releases the enzyme from the mRNA and consequently results in increased translation of DHFR protein (5–7). In addition to the described translational regulation of DHFR in cancer cells exposed to MTX, increased levels of DHFR also result through DHFR gene amplification, a common mechanism of acquired resistance to this drug (8). In contrast to rapid translational modulation of DHFR, gene amplification occurs in response to chronic exposure to antifolates, and elevated cellular levels of DHFR result from transcription of multiple DHFR gene copies.
Regulation of exogenous gene products in vivo has become increasingly important with the development of gene therapies to treat human diseases such as cancer (9, 10). We initiated the study presented here with the hypothesis that proteins of interest can be fused to DHFR and thereby adopt the cellular regulation mechanisms of the DHFR protein, thus regulating exogenous fusion proteins in a nontranscriptional manner by small drug molecules. Here, we report intracellular up-regulation of exogenous DHFR fusion gene products via antifolates in vitro and in vivo. Presented data indicate that the up-regulation is specific and translationally controlled.
Materials and Methods
Materials.
Tissue culture material was obtained from Corning. Enzymes were purchased from NEB or Stratagene. The anti-enhanced green fluorescent protein (EGFP) antibody was from Roche Molecular Biochemicals, the anti-β-actin antibody was from Sigma, and the secondary antibodies were from Santa Cruz Biotechnology. All DNA primers and probes were obtained from Keystone Laboratories (Menlo Park, CA). Antifolates were purchased from U.S. Bioscience (TMTX; West Conshohocken, PA) or Immunex (MTX), and ganciclovir (GCV) was from Roche Clinical Laboratories, Burlington, NC. [124I]2′-fluoro-2′-deoxy-5-iodouracil-β-d-arabinofuranoside (FIAU) was synthesized by the Preparative Synthesis Chemistry and Radiochemistry/Cyclotron Core Facilities at Memorial Sloan–Kettering Cancer Center (MSKCC).
Tissue Culture.
Colon cancer cells (HCT-8, HCT-116) were cultivated in RPMI medium 1640 supplemented with 100 units/ml penicillin/100 μg/ml streptomycin/10% dialyzed FBS (dFBS; Atlanta Biologicals, Norcross, GA). The AM12 and the NIH 3T3 cells were cultivated in DMEM containing high glucose (4.5 mg/ml), penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% dFBS. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 and regularly checked for mycoplasma contamination.
Plasmids.
All retroviral vectors used are based on the Moloney murine leukemia virus-based (SFG) plasmid described (11). The coding sequence for a double mutant (Phe-22–Ser-31) DHFR has been described (12). Use of the internal ribosome entry site (IRES) element was reported before (13). Published coding sequences for EGFP (accession no. U55761) and herpes simplex virus 1 (HSV1), thymidine kinase (TK) (accession no. AB009258) were used. Recombinant DNA manipulations were verified by DNA sequencing.
Transfections and Retroviral Infections.
Transfection.
Transfections of parental GP+envAM12 cells (14) with EGFP-containing plasmids were carried out by using DOTAP transfectant reagent {N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; Roche Molecular Biochemicals}.
Retroviral infection.
Retrovirus-producing cell lines were generated by transfection of the DHFR-HSV1 TK plasmid in parental GP+envAM12 cells. Transfection was performed three times at cell densities of 30%, 50%, and 75% using Superfect (Qiagen, Chatsworth, CA) as described by the manufacturer. Clones of retrovirus-producing cells were selected in 150 nM TMTX. Viral titers were determined against NIH 3T3 cells. Retrovirus-producing AM12V cells were grown in medium without drug to a density of 60–80%; the day before the infection, medium was changed. Viral transduction was performed by infecting colon cancer cells plated 48 h before infection in 10-cm dishes. Four single virus exposures with the desired multiplicity of infection (moi), each of 6 h duration, were carried out. For each single exposure, fresh AM12V supernatant was filtered through a 0.45-μm cellulose acetate filter and supplemented with polybrene (8 μg/ml). Likewise, the 2-h viral transduction was performed in 6-well plates.
Standard and Quantitative Reverse Transcriptase (RT)-PCR.
Standard PCR.
The transgene was amplified by using specific primers (forward primer: 5′-ACATTTGCACTGGTAAACTTCATCA-3′, reverse primer: 5′-CTCAAGGAACCTCCACAAGGAG-3′), Herculase polymerase (Stratagene) and the GeneAmp 9600 (Perkin–Elmer/Cetus) thermocycler at T1, 94°C, 120 sec; T2, 63°C, 30 sec; T3, 68°C, 60 sec (1 cycle); and T1, 94°C, 30 sec; T2, 63°C, 30 sec; T3, 68°C, 60 sec (33 cycles).
QRT-PCR.
The PCR reaction was performed by using a TaqMan 1,000 RXN Gold with Buffer A kit and the ABIPrism 7700 thermocycler (Applied Biosystems). The temperature profile was 95°C, 10 min (1 cycle), and T1, 95°C, 15 sec; T2, 60°C, 60 sec (42 cycles). Sequences for primers and probes were DHFR-HSV1 TK: forward primer, 5′-GGAGGAGAAAGGCATTAAGTACAAA-3′; probe, 5′-6FAM-ATCTGGTTCCGCGTGGATCCCTC-TAMRA-3′; reverse primer, 5′-CGCAGACGCGTGTTGATG-3′. β-actin : forward primer, 5′-TGAGCGCGGCTACAGCTT-3′; probe, 5′-FAM-ACCACCACGGCCGAGCGG-TAMRA-3′; reverse primer, 5′-TCCTTAATGTCACGCACGATTT-3′.
Western Blotting.
Western blotting was done at room temperature with TBS buffer containing 0.1% Tween 20 and 5% dry nonfat milk powder, according to standard protocols and manufacturer instructions. Equal protein loading was controlled by PonceauS staining and Western blotting by using anti-β-actin antibody. The enhanced chemilumiscence reagent (Amersham Pharmacia) was used according to the manufacturer's instructions to detect the secondary antibody on the blot. If required, blots were stripped two times for 30 min in 200 mM glycine (pH 2.2)/0.1% SDS/1% Tween-20 at room temperature. Measurement of signal intensities was carried out with an Alpha Imager (Alpha Innotech, San Leandro, CA) and ALPHA EASE 5.1 software.
Treatment of Cells with Drugs and Preparation of Lysates.
Drug treatment.
Compounds used in this study were dissolved in sterile water (TMTX, DHF) or DMSO (cycloheximide, actinomycin D). For chronic TMTX treatment, cells were treated in three cycles with medium containing increasing concentrations of TMTX (25 nM, 50 nM, and finally 100 nM) for 9 days in each cycle. For short time drug treatment, cells in log phase were cultured in 25-ml of medium in 15-cm dishes.
Cell lysates.
Preparation of cell lysates was performed at 4°C. Cells were washed with PBS, collected by centrifugation (1,000 × g, 1 min) and resuspended in TBS buffer containing 200 μM PMSF, 1.5 μg/ml aprotinin and 10 mM β-mercaptoethanol. After two sonications (30% output, Vibra Cell, Sonics & Materials, Danbury, CT) for 10 s each cycle, the lysate was centrifuged (14,000 × g, 25 min), and the supernatant was immediately used or stored at −20°C.
Tumor lysates.
Tumor tissue was pulverized in liquid nitrogen, weighed, suspended in a volume equal to 10× the tumor weight of SDS-PAGE sample buffer without glycerol and dye, and boiled for 5 min. Lysate was centrifuged at 14,000 × g for 25 min, and the supernatant was immediately used or stored at −20°C.
Cytotoxicity Assays.
Colony formation assay.
Cells (1 × 103) were plated in a volume of 12 ml of medium with or without drug (20 μM GCV) for 12 days in 10-cm dishes. Cells were stained with crystal violet solution, and colonies with a size of at least 0.5 mm were counted.
XTT assay.
In each well of a 96-well plate, 1.5 × 103 cells were plated in 300 μl of medium containing the desired concentration of drug, and after 5 days, the XTT assay was performed according to standard protocols. Absorbance was measured in a microplate reader (EL 340, Bio-Tek, Burlington, VT) at a wavelength of 450 nm (cut off = 630 nm).
Xenograft Tumor Model.
All work with animals was carried out according to institutional guidelines under the auspices of an animal protocol approved by the Institutional Animal Care and Use Committee at MSKCC. Male athymic (RNU) rats, 6–8 weeks old, were obtained from National Institutes of Health and housed in an MSKCC animal facility. To generate a flank tumor model, rats were anesthetized with 3 ml/kg of a 1:2 mixture Ketaset (100 mg/ml, Fort Dodge Animal Health, Overland Park, KS) and Aceproject (10 mg/ml, Vetus Animal Health, distributed By Burns Veterinary Supply, Farmers Branch, TX) given i.p. and subsequently injected s.c. with 1 × 106 cells in 100 μl of serum-free medium.
Fluorescent Microscopy and Positron-Emission Tomography (PET) Imaging.
Fluorescent microscopy.
Cells transfected with EGFP-expressing vectors were cultured for at least 12 days and subsequently sorted for EGFP-positive cells by using a FACS Vantage SE cell sorter (Becton Dickinson). For fluorescent microscopy, these cells were grown in T25 flasks. Confluent cells were cultured in fresh media supplemented with or without TMTX. Fluorescent images of randomly selected areas of the monolayer were taken with a Zeiss Axiovert S 100 microscope.
PET imaging.
Each rat received 2 ml of 0.9% NaI 48 h before imaging. The [124I]FIAU (260 μCi per animal; 1 Ci = 37 GBq) was injected under anesthesia into the rat penile vein 24 h before PET scanning. Sterile water was injected i.p. the day of imaging to enhance tracer clearance. Animals were sedated in the PET suite as described above and placed in a specially designed animal holder in the PET camera field. Imaging was performed with a GE Advanced PET tomograph (General Electric). The durations of transmission and emission scans were 7 and 20 min, respectively.
Immediately after the imaging session, the animals were killed, and tumor samples were weighed and assayed for [124I]FIAU radioactivity by using a Packard 5500 γ spectrometer (Packard).
Results
Specific and Time-Dependent Increase of DHFR-EGFP Fusion-Protein Levels in Mouse Cells Exposed to Trimetrexate.
To determine if up-regulation of DHFR fusion proteins occurs in cells exposed to TMTX, we first generated a retroviral vector encoding for a DHFR-EGFP fusion protein (Fig. 1). Transfection of the vector containing the fusion DHFR-EGFP into mouse cells (AM12) resulted in DHFR-EGFP fusion-protein expression (Fig. 2A). Increased cellular DHFR-EGFP fusion-protein levels were detected at 24 and 48 h after treatment with 1 μM of the antifolate trimetrexate (Fig. 2A). We next assessed whether the observed up-regulation of EGFP was specific for the fusion protein. Mouse cells were transfected with a vector encoding either for DHFR and EGFP separated by an IRES element or a vector containing only EGFP. These two nonfusion constructs did not result in an increase in EGFP levels in response to TMTX treatment (Fig. 2 B and C). Next, DHFR-EGFP fusion protein-expressing cells were exposed for 48 h to TMTX. After 24 h TMTX exposure, the cells were treated with cycloheximide, resulting in a reduction of the translational up-regulation, whereas addition of the transcription inhibitor actinomycin D had no effect, as determined by Western blotting (Fig. 2D).
Figure 1.
Schematic representation of the retroviral vectors used. The retroviral vectors code for (A) a DHFR (double mutant Phe-22-Ser-31)-EGFP fusion protein, (B) DHFR and EGFP separated by an IRES element, (C) EGFP or (D) a DHFR-HSV1 TK fusion protein. Transgene expression is controlled by the retroviral 3′-long terminal repeats (LTR) and enhanced by a myeloproliferative sarcoma virus (MPSV) element (▾). Unique restriction sites are indicated. ψ, packaging signal.
Figure 2.
Specific increase of DHFR-EGFP fusion-protein levels in mouse cells exposed to TMTX. AM12 cells expressing DHFR-EGFP (A), DHFR-IRES-EGFP (B), or EGFP (C) were exposed to 1 μM TMTX for 24 and 48 h and subsequently analyzed for EGFP protein levels by Western blotting. (D) AM12 cells expressing DHFR-EGFP were treated with TMTX for 48 h. After 24 h, samples were exposed to cycloheximide (50 μg/ml) or actinomycin D (1 μg/ml). Cellular DHFR-EGFP levels were determined by Western blotting using an anti-DHFR antibody. (E) DHFR-EGFP-expressing AM12 cells were exposed to 1 μM TMTX (control, water) for 48 h. After 24 (data not shown) and 48 h, cell fluorescence was monitored with fluorescent microscopy. Pictures were taken with the same settings, first from the TMTX-treated cells, then followed by the control cells.
In a subsequent set of experiments, fluorescent microscopy was used to visualize directly the effect of TMTX on cellular DHFR-EGFP levels in living cells. Fig. 2E shows that the DHFR-EGFP fusion protein is functionally active, and fluorescence increased over time in DHFR-EGFP-expressing cells treated with TMTX compared with controls. Also, cells expressing DHFR and EGFP as single proteins did not show an altered EGFP expression in response to TMTX treatment (data not shown).
Increase of DHFR-HSV1 TK Fusion-Protein Levels in Colon Cancer Cells Exposed to Antifolates.
To provide further evidence for an antifolate-induced DHFR fusion gene up-regulation, we studied human colon cancer cells retrovirally transduced to express a fusion protein of DHFR and HSV1 TK (15). Bulk transduction of HCT-116 and HCT-8 cells was carried out with an moi of 30 for 24 h or an moi of 2.5 for 2 h. This resulted in the cell lines 116HT (moi 30, 24 h) and 116LT (moi 2.5, 2 h) as well as 8HT (moi 30, 24 h) and 8LT (moi 2.5, 2 h). Successful gene transfer and transgene expression in these cell lines were confirmed by PCR (Fig. 3A) and Western blotting (Fig. 3B), respectively. The percentage of infected cells was determined by colony formation assay using the GCV sensitivity of DHFR-HSV1 TK-expressing cells as follows: 116HT, very high (no colony formation); 116LT, 80%; 8HT, 53%. Infection of 8LT cells was not detectable with the assay used. Next, we measured GCV and TMTX cytotoxicities of the transduced cells. The 116LT cells exhibit a moderate and 116HT cells exhibit a higher GCV sensitivity and TMTX resistance as compared with the parental cell line (Fig. 4 A and C). This finding correlates with the observed levels of fusion-protein expression in these cells (Fig. 3B). In a similar way, detected fusion-protein expression correlates with the drug cytotoxicity in transduced HCT-8 cells. 8HT cells, but not 8LT, cells showed GCV sensitivity and TMTX resistance (Fig. 4 B and D). In summary, HCT-8 cells showed substantially lower transduction and transgene expression in comparison to HCT-116 cells under comparable transduction conditions and correlated with lower levels of GCV sensitivity and TMTX resistance.
Figure 3.
Retroviral transfer and expression of the DHFR-HSV1 TK fusion gene in colon cancer cells. (A) Detection of the fusion gene in transduced HCT-8–116 cells by PCR. The transgene-specific 860-bp fragment was amplified from a genomic DNA template. Mock, mock-transduced control cells. (B) Western blot analysis using an anti-DHFR antibody.
Figure 4.
GCV sensitivity and TMTX resistance of parental, transduced, and TMTX-exposed colon cancer cells. GCV and trimetrexate sensitivity of parental and transduced HCT-116 and HCT-8 cells were measured with an XTT assay. Cell line abbreviations: 8LT, low-transduced HCT-8 cells; 8HT, high-transduced HCT-8 cells; 8LT-CT, low-transduced and antifolate treated HCT-8 cells; 116LT, low-transduced HCT-116 cells; 116HT, high-transduced HCT-116 cells.
When HCT-8 cells were treated chronically with increasing concentrations of TMTX, a 250-fold increase in GCV sensitivity (IC50) was measured (by XTT-assay; Fig. 4B), accompanied by an increased gene copy number (data not shown) and fusion-protein expression in the 8LT-CT cells (Fig. 3B and Fig. 5). Thus, amplification of the fusion gene and increased expression of HSV1 TK occurred with chronic antifolate treatment.
Figure 5.
Increased DHFR-HSV1 TK fusion-protein levels in various transduced and parental colon cancer cells exposed to antifolate for 24 h. Transduced and parental HCT-8 and HCT-116 cells were exposed to 1 μM TMTX or MTX for 24 h. Shown is a Western blot analysis using an anti-DHFR antibody.
In another set of experiments, the retrovirally transduced HCT-116 and HCT-8 cells were exposed for 24 h to TMTX. Increased DHFR-HSV1 TK levels were observed in all transduced cell lines (Fig. 5). However, the fold increase upon TMTX exposure was cell-line dependent. The 8HT cells showed a moderate increase, 116LT cells a higher increase, and 116HT cells an even higher increase in fusion-protein levels. When the chronically TMTX-treated 8LT-CT cells were allowed to grow in the absence of drug, a significant induction of the fusion protein occurred after a subsequent new exposure to TMTX. To demonstrate that the fusion protein contained DHFR, an anti-DHFR antibody was used. As expected, an increase of endogenous DHFR levels, as well as the exogenous DHFR-HSV1 TK in response to TMTX treatment, was noted. Moreover, the amount of increase of the endogenous DHFR was comparable to the fusion-protein increase. The observed increase in DHFR-HSV1 TK or DHFR levels in transduced cells was similar to the DHFR increase in untransduced, parental HCT-116 and HCT-8 cells.
To show dose dependence of DHFR-HSV1 TK induction, 8LT-CT cells were exposed to increasing concentrations of TMTX or MTX. Concentration-dependent induction of DHFR-HSV1 TK was noted with both antifolates (Fig. 6). Interestingly, increased fusion-protein levels also were seen after treatment of 8LT-CT cells with high concentration of the DHFR substrate dihydrofolate. In contrast, treatment of cells with etoposide, a topoisomerase II inhibitor, did not cause induction of the fusion protein (data not shown).
Figure 6.
Dose-dependent increase of DHFR-HSV1 TK fusion-protein levels in colon cancer cells exposed to antifolate. Western blot analysis using an anti-HSV1 TK antibody to detect the fusion protein. DHFR-HSV1 TK and β-actin Western blot signals were quantified, and the DHFR-HSV1 TK signal intensity relative to the β-actin signal intensity was calculated. These values are represented by the bar graph.
To confirm that the increase in DHFR was related to translational regulation, the levels of DHFR-HSV1 TK mRNA before and after TMTX, MTX, or DHF treatment were measured. As shown in Table 1, no alterations of the fusion-protein mRNA expression in response to the drug exposure were detected, indicating that the increase was caused by relief of inhibition of DHFR translation by these folate compounds and/or protection of DHFR from degradation, consistent with previous studies (4–6).
Table 1.
Fusion protein mRNA expression
| Cells | Relative expression DHFR-HSV1 TK mRNA |
|---|---|
| 8LT-CT | 3.7 ± 1.1 |
| 8LT-CT, 1 μM TMTX, 24 h | 4.7 ± 1.4 |
| 8LT-CT, 1 μM MTX, 24 h | 3.6 ± 0.8 |
| 8LT-CT, 50 μM DHF, 24 h | 3.5 ± 0.9 |
DHFR-HSV1 TK Levels Increase in Transduced Tumor Xenografts After TMTX Treatment.
Encouraged by the described in vitro observations, the in vivo relevance of the DHFR-HSV1 TK induction by TMTX was addressed. Rats bearing flank tumors derived from 8LT-CT cells were treated with 10 mg/kg TMTX for 3 days or a single high dose of 100 mg/kg. The level of DHFR-HSV1 TK fusion protein in the tumor tissue of antifolate-treated animals, as well as water-treated rats, was analyzed. All antifolate-treated animals showed elevated levels of the fusion protein ranging from 1.5 to 4-fold compared with controls (Fig. 7), and the mean increase was at least 2-fold. The second set of experiments used rats bearing tumors derived from 8LT-CT and parental HCT-8 cells and in vivo imaging. Before the imaging in living rats, animals received three cycles of the three times daily doses of TMTX. By using PET and the tracer [124I]FIAU (16), an increase in tumor-signal intensity in TMTX-treated rats as compared with control rats was observed (Fig. 8). Measurements of [124I]FIAU uptake (% dose per g) from seven treated and seven control animals showed a 2.6-fold higher [124I]FIAU accumulation in transduced tumor tissue following TMTX treatment (0.348 ± 0.175% dose per g), as compared with untreated controls (0.132 ± 0.04% dose per g).
Figure 7.
Transduced tumor xenografts show increased DHFR-HSV1 TK levels after TMTX treatment. Tumors derived from 8LT-CT cells were grown in 18 RNU rats to an average size of 560 mm3 (range 90–1,100 mm3). Subsequently, six animals each were treated with either 10 mg/kg TMTX for 3 days (■) or 100 mg/kg TMTX (▴) given in two 50 mg/kg doses the day before tumor sampling. Six control animals received water (□). Tumors were analyzed ex vivo for cellular DHFR-HSV1 TK levels by immunoblotting with an anti-DHFR antibody and, subsequently, after striping of the membrane with an anti-β-actin antibody. Western blot signal intensities were measured and the fusion-protein signal intensity relative to the β-actin signal was calculated. These values are shown; bold numbers indicate mean values including SD.
Figure 8.
TMTX treatment results in increased FIAU accumulation in transduced tumor xenografts in vivo. Shown are digital pictures as well as axial and transaxial tumor PET scans obtained from (A) an antifolate-treated and (B) water-treated (control) RNU rat. The inserted scans show the heart as control.
Discussion
The ability to control levels of exogenous, therapeutic proteins in cells is an important step toward progress in molecular therapy. Here, we use a unique drug-response mechanism of the highly regulated enzyme dihydrofolate reductase to increase cellular levels of exogenous proteins both in vitro and in vivo.
Thus far, the exact mechanism of this rapid “induction” of DHFR after exposure of cells to antifolates has not been described in detail. However, studies provide evidence that this cellular protein increase is caused by a relief of translational autoregulation of DHFR by MTX or TMTX (1–7, 17). Here, we show that antifolates are able to increase cellular levels of fusion proteins, e.g., DHFR-EGFP and DHFR-HSV1 TK in mouse and colon cancer cell lines, respectively (Figs. 2 and 5). Thus, the induction is independent of both the cell type and the protein fused to DHFR. However, as shown in Fig. 5, the intensity of induction measured after 24 h varied. Importantly, only DHFR or DHFR fusion proteins showed this increase. Cells transfected with DHFR-IRES-EGFP showed a barely detectable increase in EGFP levels upon exposure to antifolates (Fig. 2B). Furthermore, by transfecting cells with SFG vector containing EGFP and subsequent TMTX treatment of the cells we ruled out a nonspecific effect of the SFG vector backbone (Fig. 2C). In addition, and in accordance with earlier studies, we observed a decreased cellular induction of the DHFR-EGFP fusion protein after cycloheximide treatment, whereas actinomycin D had no effect on the induction (Fig. 2D). No change in DHFR-HSV1 TK mRNA levels after exposure to antifolates was detected (Table 1), as was observed previously (4).
Interestingly, the other mammalian enzyme known to exhibit induction of the protein by the substrate, cofactor, or inhibitor is thymidylate synthase (TS), a protein catalyzing the NADPH-dependent conversion of dUMP and THF to dTMP and DHF. Thus, TS, like DHFR, is a component of the cell cycle-dependent and highly regulated de novo dTMP production, which is essential for DNA synthesis. Similar to DHFR regulation, TS has been reported to specifically bind to its own mRNA and hence inhibit its own translation (18, 19). A recent study raises the possibility that binding of FdUMP to TS protects this enzyme from degradation and also could be responsible for the observed increased TS levels after fluoropyrimidine exposure (20). In contrast to DHFR, TS is larger, functional as a dimer, and demonstrates only a 2- to 4-fold induction in patients (21). Thus, DHFR appears to be more suitable than TS for the generation of inducible functional fusion proteins.
Previous studies with another DHFR fusion protein (22) and the findings presented here indicate that DHFR fusion-protein activities are fully functional (Figs. 2E, 4, and 8), and that the fusion-protein induction is similar to the induction of the endogenous DHFR (Fig. 5). We used a double-mutant DHFR (12) to protect transduced cells from antifolate toxicity.
In this study, we describe a nontranscriptional, fusion protein-based regulation of an exogenous gene product by a small molecule. Alternate strategies to regulate cellular protein levels by controlling gene expression involve transcriptional regulation. The potential of doxycycline-responsive gene expression was demonstrated recently in a mouse model of a neurodegenerative disease (23), in which the successful transfer and tightly controlled expression of tyrosine hydroxylase in brain grafts of human neural progenitors was reported. Enzyme expression depended on the presence of doxycycline. Another system used the immunosuppressive drug rapamycin to mediate the reconstitution of a functional transcription factor and thus transgene expression. Like the first system, this system uses a single-vector coding for the essential control elements, as described (24). Both described transcriptional regulations show induction of gene expression of approximately four orders of magnitude in vitro. However, they require vectors containing relatively long sequences, which encode for at least three transcriptional control elements. In contrast, the approach presented in this study is based on the fusion of the sequence encoding the protein of interest to the 560-bp DHFR sequence. It might not be possible to express all proteins as functional DHFR fusion proteins; however, there is evidence that the relatively small DHFR protein is an appropriate partner in fusion proteins; e.g., DHFR fusion proteins have been found to occur in nature in protozoa such as Plasmodium, which expresses a DHFR-TS fusion protein. Moreover, a number of artificial fusion proteins have been successfully generated. These fusion proteins were used for protein purification strategies using MTX affinity chromatography or gene therapy approaches to confer drug resistance to antifolates and to cytosine arabinoside, using a DHFR-cytidine deaminase (CD) fusion cDNA (22). Identification of the minimal region of DHFR mRNA that is essential for antifolate-mediated regulation may improve in vivo regulation of the second gene of interest.
Retroviral gene transfer delivery is known to be critical for in vivo applications. We used a retroviral vector in this study because the gene transfer method provides specificity for dividing cells but, compared with adenoviral delivery, lower transduction efficiencies. In contrast to most gene therapy studies, we used bulk transduced cell lines and not clonal cell lines in this study. Bulk transduction may reflect retroviral infection in vivo better than clonal cell line-derived tumors. We observed significantly different transduction efficiencies between HCT-8 and HCT-116 cells, which resulted in distinct levels of fusion-protein expression and cytotoxicity characteristics. Both cell lines are human, primary colon cancer cell lines but may display different levels of retroviral receptors. Taken together, our observations point to a significantly cell type-dependent retroviral infection efficiency. Chronic exposure to increasing concentrations of TMTX resulted in a significantly increased GCV-sensitivity of the low-transduced 8LT cells along with detectable levels of DHFR-HSV1 TK fusion protein and increased copies of the corresponding transgene and mRNA. We were able to increase further fusion-protein levels by stepwise exposure up to 800 nM TMTX (data not shown). These observations were likely caused by a selection for transduced cells.
Here, we used HSV1 TK, the tracer [124I]FIAU, and PET scanning to image a DHFR-HSV1 TK fusion protein in transduced tumors in vivo. Recently, a study described the feasibility of noninvasive imaging of a CD-HSV1 TK fusion protein (25). In this study, we demonstrate increased levels of the DHFR-HSV1 TK fusion protein in transduced tumors after antifolate treatment. Additional studies that evaluate the ability to increase PET signal or fluorescence intensities of tumors transduced with DHFR-HSV1 TK or DHFR-EGFP, respectively, in tissue of living animals by antifolate treatment will be of interest. A further application of increased cellular levels of therapeutic polypeptides such as HSV1 TK by induction as DHFR fusion protein might be an increased cellular response to therapies such as GCV because of higher cellular levels of the therapeutic protein after antifolate treatment.
In summary, we have taken advantage of a cellular, adaptive drug-resistance mechanism, i.e., a post translational, fusion protein-based strategy to regulate the level of exogenous proteins in vitro and in vivo. The observations presented here should be useful for application in gene therapy, gene expression, and drug resistance research.
Acknowledgments
The HSV1 TK antibody was a kind gift of Dr. W. C. Summers (School of Medicine, Yale University, New Haven, CT). This work was supported by National Institutes of Health Grants CA 08010 and CA 61586 (to J.R.B.) and CA 86438-02 (to D.B.). The Dr. Mildred Scheel Foundation for Cancer Research generously supported P.M.-K.
Abbreviations
- DHFR
dihydrofolate reductase
- MTX
methotrexate
- TMTX
trimetrexate
- moi
multiplicity of infection
- RT
reverse transcriptase
- GCV
ganciclovir
- EGFP
enhanced green fluorescent protein
- PET
positron-emission tomography
- FIAU
2′-fluoro-2′-deoxy-5-iodouracil-β-d-arabinofuranoside
- HSV1
herpes simplex virus 1
- IRES
internal ribosome entry site
- TK
thymidine kinase
- TS
thymidylate synthase
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