CRIM1 is Expressed at Higher Levels in Drug-resistant than in Drug-sensitive Myeloid Leukemia HL60 Cells

Materials and Methods

Cell lines and culture conditions. Human leukemia HL60 cells that are highly sensitive to doxorubicin (HL60 S), and the sub-cell line HL60 R0.5, which are resistant to 0.5 μM doxorubicin (11), were grown in suspension in Roswell Park Memorial Institute (RPMI) 1640 medium (without phenol red) (GIBCO, Invitrogen Co., Paisley, Scotland, UK), containing 10% heat-inactivated fetal bovine serum (GIBCO) and 2.5% 1 M Hepes (GIBCO). Cells were cultured at 37°C in a humidified atmosphere of 5% CO₂ in air. Resistant cells were maintained without drug for two weeks prior to each experiment.

Exposure of cells to cytostatic drugs. Cells were suspended in pre-heated medium (37°C) in culture flasks at a concentration of 5×10⁵ cells/ml and a final volume of 8 ml. Each cell line was exposed to a final concentration of either 0.2 μM DNR (Cerubidin; Aventis Pharma, Bromma, Sweden), 0.5 μM Ara-C (Cytarabine; Pfizer AB, Sollentuna, Sweden) or RPMI 1640 as a negative control, in three separate experiments. These concentrations were chosen to mimic clinically relevant exposure concentrations (12). The reaction was stopped with 10 ml of ice-cold RPMI 1640. Cells were collected by centrifuging at 400 × g for 5 min at 4°C and the supernatant discarded. For each experiment, samples were collected before drug exposure and after 2 and 16 hours of drug exposure. Incubations were performed twice, each time in duplicate. From the first incubation, first-strand cDNA synthesis was carried out twice; hence real-time RT-PCR was performed a total of three times.

RNA preparation and cDNA synthesis. Total RNA was isolated with an RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Briefly, RNA from approximately 4×10⁶ cells was eluted in 50 μl of RNase-free water and stored at −80°C until analysed. The total RNA concentration and the quality of the isolated RNA were determined using an Agilent Bioanalyzer 2100 and an RNA 6000 Nano Assay Kit (Agilent Technology, Santa Clara, USA) according to the manufacturer's instructions. The quality of the RNA was determined using the ratio of 28S to 18S. A ratio above 1.5 was used as a cut-off for acceptable quality RNA. For first-strand cDNA synthesis a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used according to the manufacturer's instructions. Briefly, 0.5 μg of total RNA was used to produce first-strand cDNA with dNTP in a final volume of 20 μl in RNase-free water and stored at −20°C until analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).

Rreal-time RT-PCR. Real-time RT-PCR was performed using the thermal cycler TaqMan 7500 Fast Real-Time PCR System (Applied Biosystems) and the 7500 Fast Sequence Detection and Absolute Quantification software packages. PCR cycling conditions were: step one, 95°C for 2 min and step two, 95°C for 3 s and 60°C for 30 s (step two repeated 40 times). As an internal control, human placenta DNA was used and as house-keeping gene 18S (Hs99999901_s1 from Applied Biosystems) was used. PCR reactions were performed in 15 μl using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and 1.5 μl cDNA. Experiments were carried out in duplicate and the mean values used for analysis.

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Figure 1.

CRIM1, SMAD5 and BMP4 mRNA levels in drug-sensitive and resistant cell lines. When baseline CRIM1, SMAD5 and BMP4 mRNA levels were set to 1 in drug sensitive HL60S cells, CRIM1 mRNA levels before drug exposure were 15-fold higher in drug-resistant HL60 R0.5 cells. SMAD5 mRNA levels were 8-fold higher in drug-resistant HL60 R0.5 cells. BMP4 mRNA levels in HL60 R0.5 before drug exposure were 3.4-fold higher compared to HL60S.

Immunofluorescence. Drug sensitive and drug-resistant HL60 cells, at a concentration of 5×10⁴ cells in 50 μl, were allowed to dry on glass cover slips, in air at room temperature, overnight. When dry, cells were fixed with cold methanol (−10°C), and air dried for 5 min at room temperature. After rinsing three times in PBS with 0.1% saponin (Sigma Aldrich, St. Louis, MO, USA) cells were washed twice in PBS, each time for 5 min. To minimize nonspecific binding, all samples were blocked for 30 min in 1.5% bovine serum albumin (BSA) blocking solution (Sigma Aldrich) diluted in PBS plus 0.1% saponin. Samples were incubated with a primary polyclonal rabbit antibody against CRIM1 (Atlas antibodies, Stockholm, Sweden) overnight at 4°C, at a dilution of 1:200 in PBS plus 1.5% BSA blocking solution. After washing 3×5 min with PBS, samples were incubated with a FITC-conjugated secondary antibody (1 μg/ml) (Dako, Glostrup, Denmark) diluted in PBS with 1.5% BSA blocking solution for 30 min. The cells were counter stained using DAPI mixed with mounting media and the slides were mounted. Samples were analyzed using a Leica Confocal Systems Microscope equipped with a Leica confocal system (Leica Microsystems, Heidelberg, GmbH, Germany).