The radiosensitising effect of gemcitabine and its main metabolite dFdU under low oxygen conditions is in vitro not dependent on functional HIF-1 protein

The human tumour cell lines included were MDA-MB-231 (breast adenocarcinoma; wild type (wt) HIF-1) and the sublines MDA-MB-231 dnHIF (dominant-negative HIF-1α; HIF-1 activity inhibited) and MDA-MB-231 empty vector control (EV; functional HIF-1). MDA-MB-231 sublines were constructed as described previously [16], resulting in MDA-MB-231 cells stably expressing dnHIF tagged with enhanced green fluorescence protein (eGPF) or eGFP alone (MDA-MB-231 dnHIF and MDA-MB-231 EV, respectively). The dnHIF construct inhibits HIF-1 activity by competing with endogenous HIF-1α for interaction with HIF-1β and DNA binding; it is however likely that non-canonical regulation by HIF-1 is not inhibited, since the dnHIF construct is identical to endogenous HIF-1α except for loss of the oxygen-dependent degradation domains and DNA-binding domains. All cell lines were free from mycoplasma contamination. Cultures were maintained in exponential growth in a humidified 5% CO₂/95% air atmosphere at 37°C (normoxia).

Hypoxia (<0.1% O₂) was achieved in a Bactron IV anaerobic chamber (Shel Lab, Cornelius, USA), as described previously [17]. Hypoxic incubation was initiated after cells had been cultured under normoxia overnight, allowing attachment to culture dishes.

Cells were placed under normoxia or hypoxia for 18 h, yielding a robust induction of the expression of HIF-1α and HIF-1-induced downstream targets. Subsequently, cells were lysed and protocols were used as previously described [18]. In short, cells were lysed in 100 μl lysis buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100 v/v, 0.5% Nonidet P-40 v/v, 2 mM leupeptin, 0.15 mM aprotinin, 1.46 mM pepstatin, 1 mM phenylmethansulfonyl fluoride). For western blot analysis, proteins (20 μg/lane) were resolved on a 7.5% SDS-PAGE gel and electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Schwalbach, Germany) using standard procedures. After blocking with 5% non-fat dry milk w/v in PBS-T (137 mM NaCl, 2.7 mM KCl, 4.3 mM di-sodiumhydrogenphosphate, 1.4 mM potassium-di-hydrogenphosphate, 0.1% Tween20 v/v) overnight at 4°C, the blot was probed with primary antibodies (mouse monoclonal anti-HIF-1α (BD Transduction Laboratories, Oxford, UK); monoclonal anti-CA9 (clone M75; kindly provided by Dr. Jaromir Pastorek, Bratislava, Slovakia)). The blot was then reacted with suitable secondary alkaline phosphatase-conjugated antibodies (Dianova, Hamburg, Germany), followed by detection of the protein with CDP-Star chemiluminescent reagent (Applied Biosystems, Foster City, USA). Finally, the blot was stripped and reprobed for β-actin (mouse monoclonal anti-β-actin, Sigma-Aldrich, Dorset, UK) to ensure equal loading and transfer of proteins.

To assess dnHIF-mediated inhibition of CA9, cells were cultured on sterile coverslips under normoxia or hypoxia for 18 h, fixed for 10 min and permeabilised. Cells were incubated with a mouse monoclonal anti-human CA9 antibody (clone M75) for 1 h at 37°C, washed and incubated with a secondary Alexaflour antibody (Life Technologies, Paisley, UK) for 45 min at 37°C. Cells were then washed and nuclei were counterstained with 4’ ,6-diamidino-2-phenylindole (DAPI) and mounted in DAKO fluorescent mounting media (DAKO, Cambridgeshire, UK). Relative localisation of green (eGFP; to identify eGFP in empty vector cells and the dnHIF construct in dnHIF cells), red (for CA9) and blue (for nuclei) fluorescence was analysed with a snapshot wide-field fluorescence microscope and MetaView software.

HIF-1α activity was assessed as described previously [18]. Briefly, cells were transfected with an adenovirus containing trimers of the LDH-A HRE linked to luciferase and were exposed to hypoxia for 18 h. Afterwards, cells were lysed, luciferase activity per μg protein was calculated and activity of MDA-MB-231 dnHIF cells was normalised to MDA-MB-231 EV control cells.

Cells were incubated under normoxia or hypoxia for 18 h, then the media was removed for analysis of secreted VEGF levels as previously described [18]. The concentration of secreted VEGF was determined using a Duoset Human VEGF ELISA kit (R&D Systems, Abingdon, UK) according to the manufacturer’s instructions, and corrected to the amount of protein within the cell cultures from which the medium was taken.

After 24 h incubation under hypoxia, total RNA samples were isolated from 4.10⁶ normoxic and hypoxic cells, using RNeasy® Mini Kit (Qiagen, Venlo, The Netherlands). The concentration of extracted RNA (A₂₆₀/A₂₈₀ ratio) and purity (A₂₆₀/A₂₃₀ ratio) were measured by Nanodrop ND-1000 spectrophotometer (Isogen, Sint-Pieters-Leeuw, Belgium). The quality of isolated RNA was confirmed by capillary electrophoresis with an Agilent 2100 Bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands). One μg RNA was reversed transcribed using ReactionReady First Strand cDNA Synthesis Kit (Qiagen) in accordance with the manufacturer’s instructions. The relative expression of 84 genes related to the hypoxia signalling pathway was assessed by use of the Human Hypoxia Signalling Pathway PCR Array (Qiagen) and the RT² Real-time SYBR Green/Rox PCR Master mix kit (Qiagen).

MDA-MB-231 cells were exposed to normoxia or hypoxia and treated with 0–8 nM gemcitabine or 0–4 μM dFdU for 24 h immediately before and during irradiation (0–8 Gy, room temperature, XRAD320 irradiator (Precision X-Ray, North Branford, USA)). Treatment schedule and concentrations of gemcitabine and dFdU were chosen based on previous results [12, 19]. To irradiate cells under hypoxia, custom-made airtight Perspex shells, preincubated in the anaerobic chamber overnight, were used [17]. Immediately following irradiation, hypoxic cells were reoxygenated and all cells were washed with drug-free medium. Unirradiated control cells were handled identically to treated cells. Following an 8-day incubation period, cells were stained with crystal violet and colonies (>50 cells) were counted as described previously [20].

Cells were incubated with 0–8 nM gemcitabine or 0–4 μM dFdU for 24 h, under normoxia or hypoxia. Cell cycle distribution was monitored according to the Vindelov method, as previously described [12]. Samples were analysed using a FACScan flow cytometer (Becton Dickinson). Histograms of DNA content were analysed using WinMDI software to determine the fractions in each phase of the cell cycle (G₀/G₁, S and G₂/M).

For irradiation experiments, survival rates were calculated as [mean plating efficiency of treated cells/mean plating efficiency of control cells] × 100%. Radiation survival curves were fitted according to the linear-quadratic model using WinNonlin (Pharsight, Mountain View, USA) with survival = exp(-αD-βD²). The following parameters were calculated: ID₅₀ (radiation dose producing a surviving fraction of 50%); SF₂ (surviving fraction at 2 Gy); and mean inactivation dose (MID). The oxygen enhancement ratio (OER) was determined by dividing ID₅₀ under hypoxia by ID₅₀ under normoxia. The dose enhancement factor (DEF) was calculated as ID₅₀ for control, untreated cells divided by ID₅₀ for treated cells.

Possible synergism between gemcitabine or dFdU and radiation was determined by calculation of the combination index (CI) using CalcuSyn software (Biosoft, Cambridge, UK). CI < 1.0, CI = 1.0 and CI > 1.0 indicated synergism, additivity or antagonism, respectively.

To analyse the PCR array data, relative changes in gene expression were calculated using the ΔΔC_t method. Based on the geNorm algorithm, four endogenous control genes were selected for normalisation. Each replicate cycle threshold (C_t) was normalised to the average C_t of the four endogenous controls on a per plate basis and mRNA expression levels were presented as fold changes.

Exposure to 18 h hypoxia induced expression of HIF-1α and its downstream target carbonic anhydrase 9 (CA9) in MDA-MB-231 wt and EV cells (Figure 1A). In MDA-MB-231 dnHIF cells, HIF-1α expression was detected in hypoxic cells exposed to reduced oxygen levels too. However, due to the presence of the dnHIF protein, CA9 expression remained markedly lower in comparison with the HIF-1α proficient cell lines. As shown in Figure 1B, the dnHIF construct was localised to the nucleus and was expressed independently of oxygen availability. Transfection with the control vector (EV) resulted in eGFP expression that was confined to the cytoplasm. Moreover, the immunofluorescence images clearly showed induction of CA9 in the EV cells under hypoxic conditions, while CA9 staining was absent in hypoxic dnHIF cells.In order to quantitatively evaluate the impact of dnHIF on HIF-1 function, an adenoviral-based HIF-1α reporter gene assay showed that HIF-activity was significantly inhibited (p < 0.01) in cells expressing the dnHIF protein (Figure 1C). Furthermore, hypoxia-induced VEGF secretion was significantly lower (p < 0.05) in dnHIF versus EV cells (Figure 1D).

Out of 84 genes related to the hypoxia signalling pathway, 11 genes showed a more than two-fold up- or downregulation in mRNA levels between normoxic and hypoxic conditions (Table 1). For HIF-1α, a decreased mRNA expression level was observed after 24 h hypoxia (Figure 2). Statistical analysis revealed no differential expression profile between MDA-MB-231 wt and EV versus MDA-MB-231 dnHIF cells for the hypoxia-related genes included in the PCR array. A significant difference in expression level under normoxia versus hypoxia was noticed for HIF-1α and angiopoetin-like 4 in MDA-MB-231 wt and EV cells (p < 0.042); in MDA-MB-231 dnHIF cells, this was only detected for angiopoetin-like 4 (p = 0.026). Significance was however lost when p-values were adjusted for multiple comparisons according to the Benjamini-Hochberg procedure.

A concentration-dependent radiosensitising effect was observed for gemcitabine and dFdU (Figure 3, Table 2), yielding a moderately synergistic to synergistic interaction between gemcitabine and irradiation. Radiosensitisation was similar under normoxia and hypoxia (p = 0.477 for gemcitabine, p = 0.563 for dFdU). Moreover, the dose enhancement factor was not significantly influenced by the cell line used (p = 0.736 for gemcitabine, p = 0.832 for dFdU) and a similar degree of radiosensitisation was observed with gemcitabine and dFdU in MDA-MB-231 wt, EV and dnHIF cells. Cell survival was significantly influenced by the concentration of gemcitabine or dFdU, dose of radiation, and oxygen tension (p ≤ 0.001). While treatment with gemcitabine or dFdU and exposure to hypoxia both had a significant impact on ID₅₀, MID and SF₂, no significant difference for these radiobiological parameters was seen between EV versus dnHIF cells.

In contrast to previous findings in other cell lines [17], exposure of MDA-MB-231 cells to hypoxia did not induce a significant increase in the percentage of G_0/1 cells (p = 0.213) (Figure 4). The number of cells in S phase was significantly influenced by the concentration of gemcitabine or dFdU (p < 0.001). Gemcitabine and dFdU caused a S phase block in MDA-MB-231 wt, EV and dnHIF cells, both under normoxia and hypoxia (Table 3). As shown on the DNA histograms (Figure 4B), the cell cycle arrest was clearly dependent on the concentration of the drug and shifted from a S phase block to an early S phase block, near the G₁/S border, with increasing concentrations of gemcitabine and dFdU. Post hoc analysis revealed no significant difference in the percentage of G_0/1, S or G₂/M phase cells between MDA-MB-231 EV and MDA-MB-231 dnHIF at any condition tested, suggesting that the cell cycle perturbations were not dependent on functionality of HIF-1α.