Cells exposed to antifolates show increased cellular levels of proteins fused to dihydrofolate reductase: a method to modulate gene expression

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.

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.

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.

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.

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.

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.