Characterization of a transport and detoxification pathway for the antitumour drug bleomycin in Saccharomyces cerevisiae

Abstract

BLM (bleomycin) is effective in combination therapy against various cancers including testicular cancer. However, several other cancers such as colon cancer are refractory to BLM treatment. The exact mechanism for this differential response of cancer cells to the drug is not known. In the present study, we created fluorescently labelled BLM-A5, which retained nearly full genotoxic potential, and used this molecule to conduct the first study to understand the transport pathway of the drug in Saccharomyces cerevisiae. Uptake studies revealed that fluoro-BLM-A5 is transported into the cell in a concentration-dependent manner. Transport of a non-saturating concentration of fluoro-BLM-A5 was modest for the first 90 min, but thereafter it was sharply induced until 300 min. The inducible transport was completely abolished by the addition of cycloheximide, suggesting that BLM-A5 uptake into the cell is dependent on new protein synthesis. Interestingly, transport of fluoro-BLM-A5 was blocked if the cells were preincubated with increasing concentrations of spermine. Moreover, a mutant lacking the Ptk2 kinase, necessary for positively regulating polyamine transport, was defective in fluoro-BLM-A5 uptake and exhibited extreme resistance to the drug. A simple interpretation of these results is that BLM-A5 may enter the cell through the polyamine transport system. We showed further that after the uptake, fluoro-BLM-A5 accumulated into the vacuole of the parent, but localized to the cytoplasm of mutants disrupted for the END3 gene required for an early step of the endocytotic pathway. In general, mutants with a defect in the endocytic pathway to the vacuole were hypersensitive to BLM-A5. We suggest that BLM-A5 is transported across the yeast plasma membrane and sequestered into the vacuole for detoxification.

Figure 1. Structure of BLM-A5 and agarose gel electrophoresis analysis of fluorescently labelled forms

(A) Depiction of BLM-A5 domains. The metal-binding domain binds to reduce iron and in the presence of oxygen forms a free radical that attacks the DNA. While the polyamine-like region is involved in DNA binding, the function of the carbohydrate moiety is unknown. This structure has been adapted from Leitheiser et al. [58]. (B, C) Resolution of fluorescently labelled BLM (F-BLM) and SPM (F-SPM) by agarose gel electrophoresis. The products formed by reacting either BLM (B, lane 2) or SPM (C, lane 2) with activated succinimidyl FITC (lane 1) were loaded on 1% agarose gel in 40 mM Mes buffer and allowed to migrate for 2 h at 50 V (see the Materials and methods section). Bands were detected by long-wavelength UV light. Open arrows indicate the loading lanes and closed arrows indicate positions of the reaction products F-BLM, F-BLM* and/or F-SPM. The * indicates the inactive form of F-BLM.

Figure 2. Purification of F-BLM by HPLC and F-BLM-induced DNA damage analysis

(A) HPLC analysis of F-BLM. F-BLM isolated from agarose gel was diluted in 0.1% TFA and subjected to HPLC using a Vydac C₁₈ column at a flow rate of 1 ml/min. F-BLM elution was monitored by reading the absorbance at 254 nm. Peaks I, II, III and IV are fractions containing F-BLM. The long arrow indicates the elution position of purified BLM-A5. (B) F-BLM-induced DNA damage. The F-BLM in peak I was tested for the ability to convert covalently closed circular DNA (form I) into the nicked DNA (form II). Lanes 1–7, 50 ng each of pBluescript plasmid incubated with 0, 0.1, 1.0 and 10 μg/ml of F-BLM (lanes 1–4 respectively) and 0.1, 1.0 and 10 μg/ml of BLM-A5 (lanes 5–7 respectively) at 25 °C for 30 min. Lane M, DNA size standard. Samples were loaded on 1% agarose gel, migrated for 1 h at 100 V, and photographed after staining with ethidium bromide.

Figure 3. F-BLM decreases cell survival and induces DNA damage

(A) Exponentially growing cells were treated with increasing concentrations of either F-BLM or the natural drug BLM-A5. Cultures were serially diluted and scored for survivors on solid YPD medium after 2 days of growth at 30 °C. (B) In vitro incorporation of [methyl-³H]dTMP by DNA polymerase I into chromosomal DNA isolated from untreated and F-BLM-treated yeast cells. Exponentially growing cells were either treated with or without 20 μg/ml of F-BLM for 2 h. Where indicated (open symbols), the chromosomal DNA was preincubated with 20 ng of purified Apn1 for 20 min before monitoring [methyl-³H]dTMP incorporation. YW465 is the parent strain (Wt) and YW778 is the mutant defective in DNA repair. Results are representative of two independent experiments.

Figure 4. Kinetics of F-BLM uptake and inhibition by CHX in the parent strain

(A) Concentration-dependent transport of F-BLM. Exponential-phase cells were incubated with increasing concentrations of F-BLM for 1 h at 30 °C, washed and the amount of F-BLM uptake was quantified using a fluorescent spectrophotometer. (B) Analysis of FITC uptake in the parent strain. (C) Time-dependent transport of F-BLM and inhibition by CHX. (B, C) Exponential-phase cells were incubated with a fixed concentration of FITC (10.0 μg/ml) and F-BLM (0.72 μg/ml) respectively, and samples were processed for drug uptake at the indicated time. CHX (10 μg/ml) was added to the cells for 30 min before the analysis of F-BLM uptake. (D) Uptake of [³H]leucine in the parent strain. Conditions for [³H]leucine uptake were the same as for F-BLM. SPM (1.0 mM for 16 h) and BLM (0.72 μg/ml for 1 h) were used to examine for interference of [³H]leucine uptake. Results are representative of three independent experiments. Wt, parent strain BY4741.

Figure 5. Inhibition of F-BLM uptake in the parent strain by SPM

Exponential-phase cells (Wt) were first incubated with increasing concentrations of SPM (0.1–1.0 mM for 16 h) followed by analysis for F-BLM uptake. Results represent the average of three independent experiments.

Figure 6. Analysis of F-BLM and [³H]leucine transport and cell survival

(A) Comparison of F-BLM uptake in the parent and ptk2Δ mutant. F-BLM uptake was analysed as in Figure 4. (B) Comparison of [³H]leucine uptake in the parent and ptk2Δ mutant. (C) Sensitivity of parent and ptk2Δ mutant to BLM. Exponentially growing cells were treated as in Figure 4. For nystatin treatment, cells were preincubated with the drug (10 units/ml for 1 h) following uptake measurements.

Figure 7. Fluorescent analysis showing F-BLM distribution in the parent and ptk2Δ mutant

(A–D) Cells were incubated with FITC (0.72 μg/ml), F-BLM (0.36 μg/ml) or F-SPM (0.36 μg/ml) for 1 h and then photographed at a magnification of 100× with a Leica fluorescent microscope equipped with a digital camera (Retiga GX 32-002TB-303). FITC and F-SPM were used as negative and positive controls respectively. (E) Identification of the vacuoles with FM4-64 dye. Vacuoles are shown with arrows.

Figure 8. F-BLM uptake and distribution, and cytotoxic effects on mutants defective in endocytosis

(A) Comparison of F-BLM uptake in the parent and various mutant strains. Cells were monitored for F-BLM transport as described in Figure 4. (B) F-SPM was used as a control to monitor SPM uptake into the parent and end3Δ mutant. F-SPM uptake was monitored as for F-BLM as described in the Materials and methods section. (C) Fluorescent analysis of F-BLM distribution in the parent and end3Δ mutant. Cells were processed for immunofluorescent microscopy as in Figure 7. (D, E) Spot-test analysis of the parent and various mutant strains for BLM and CHX sensitivity respectively. Exponentially growing parent and the isogenic mutants were serially diluted and spotted on YPD solid agar plates containing the indicated concentrations of BLM (0.45–2.0 μg/ml) or CHX (0.25 μg/ml). The plates were photographed after 2 days of growth at 30 °C. Results are representative of three independent experiments.

Figure 9. Model illustrating the transport and detoxification pathway of BLM

The drug enters the cell through a BLM transporter. The activity of the transporter might be influenced by the kinases Ptk2 and Sky1, which are known to regulate the plasma membrane polyamine transporter (see text). After uptake, BLM is channelled to the vacuole for detoxification. Interruption of the endocytic pathway to the vacuoles resulted in mutants that are hypersensitive to BLM.