Hypoxia-dependent transcriptional activation of genes is mostly governed by HIF-1α. This fact prompted us to analyze the expression patterns of HIF-1α in the SK-NF-I human neuroblastoma and CCF-STTG1 human astrocytoma cell line. To this end, cells were challenged either with 1% hypoxia or with 100 μM desferrioxamine (DFO), a hypoxia-mimicking agent that is known to stabilize HIF-1α through blocking the activity of proline hydroxylases [27]. As it can be seen in Fig. 1, the HIF-1α protein was undetectable in normoxic cultures, while both short-term hypoxic or DFO treatment dramatically upregulated the protein levels of this hypoxia dependent transcription factor. Importantly, two separate bands were recognized by the specific HIF-1α antibody that might correspond to differentially spliced variants [28]. In neuroblastoma cells, both bands were induced equally, while in CCF-STTG1 cells the staining of the upper band was more pronounced (Fig. 1).

In order to directly monitor the transcriptional activity of HIF-1α in neuroblastoma cells, we generated a luciferase reporter vector containing multiple hypoxia responsive elements inserted in the SV40 strong promoter (Fig. 2). This construct was transiently transfected into SK-NF-I cells which were subsequently subjected to 1% hypoxia or treated with DFO as described in Methods. Notably, both hypoxia and DFO treatment enhanced the activity of this reporter construct several fold over basal levels (Fig. 3). These results proved that the hypoxic signalling pathway is intact and readily inducible in SK-NF-I cells.

We aimed to study the expression of a set of dopaminergic neurotransmission specific genes under hypoxia in the SK-NF-I and astrocytoma model system. To this end, parallel cultures were maintained both in hypoxia and normoxia, and the temporal pattern of gene expression was followed by quantitative reverse transcription PCR using gene-specific TaqMan probes.

Finding a stable endogenous control gene is the cornerstone of the validation of qRT-PCR data. In order to select an optimal hypoxia-insensitive reference gene, we sought to screen for amplification efficiency and overall stability the mRNA levels of the following five candidate genes widely used in qRT-PCR studies: β-actin, hydroxymethylbilane synthase (HMBS), hypoxanthine guanine phosphorybosyltransferase (HPRT), P0 large ribosomal protein (RPLP0) and RNA polymerase II (RPII).

Table 1 shows the representative relative expression levels of these potential internal control genes measured in 8-hr hypoxic samples in SK-NF-I cells. Highly similar results were obtained in astrocytoma cells (data not presented). Based on these data, three reliable control pairs have been found (HPRT-RPLP0, HPRT-RPII and RPLP0-RPII), whereat the relative expression ratios were closest to 1. Of them, we chose the RPLP0 gene as internal control.

No significant changes in the mRNA levels of dopamine D2, D3 and D4 receptors (DRD2, DRD3 and DRD4), the dopamine transporter (DAT), monoamino oxidase A (MAOA), catechol-O-methyltransferase (COMT) and vascular endothelial growth factor (VEGF) were revealed in samples kept for 0–48 hrs in normoxia (data not shown). On the contrary, both DRD3 and DRD4 receptor mRNA levels were upregulated upon long-term (48 hrs) incubation of SK-NF-I and CCF-STTG1 cells in 1% hypoxia (Figs. 4 and 5). In neuroblastoma cells, the induction of DRD4 was much more pronounced (about eightfold compared to 48-hr untreated control levels) than that of DRD3 (Fig. 4). In contrast, the DRD3 was much more inducible than DRD4 in astrocytoma cells, and its mRNA levels were highly elevated already after 24 hr hypoxia (Fig. 5). However, the induction pattern of both receptors differed profoundly from that of VEGF, a positive control gene known to be directly activated by HIF-1α: transcription of the VEGF gene was induced already after 3 hr hypoxia and its mRNA levels dynamically increased over the entire incubation period in both cell lines.

Importantly, the DRD2 gene did not prove to be hypoxia sensitive at all in either cell lines (Figs. 4 and 5). Furthermore, no remarkable alterations were revealed in the mRNA levels of COMT, MAOA and DAT upon hypoxia in either cell lines investigated (Table 2).

DRRF (dopamine receptor regulating factor, Kruppel-like factor 16) is a zinc finger transcription factor that is considered a key regulator of post-synaptic dopamine receptors. DRRF has reportedly modulated DRD1, DRD2 and DRD3 promoter activities in a cell specific manner [29]. We were prompted to check whether activation of DRD3 and DRD4 promoters might be due to altered DRRF expression in hypoxia. It turned out that 1% hypoxia repressed DRRF levels in a time-dependent manner in SK-NF-I cells. Its concentration was the lowest after 16 hr hypoxia and then slightly elevated up to 48 hrs (Fig. 6). In conclusion, there seems to be an inverse correlation between DRD3/DRD4 and DRRF levels in the context of SK-NF-I neuroblastoma cells.

Having demonstrated the hypoxia sensitivity of the DRD3 and DRD4 genes, we aimed to examine whether elevated mRNA levels of both genes correlate well with protein expression. To this end, neuroblastoma and astrocytoma cells were cultured parallel for 48 hrs in normoxia or in 1% hypoxia, respectively, then fixed and immunostained with specific anti-DRD3 and anti-DRD4 antibodies. Cells were homogenously stained with marked cortical enrichment (Fig. 7), a pattern characteristic of membrane surface receptors [30]. We quantified staining intensities of corresponding normoxic and hypoxic samples by subjecting 500-500 cells to densitometry; however, no significant changes were revealed (data not shown). One can conclude that the transcriptional activation of DRD3 and DRD4 genes was not followed by elevation of their protein levels in these two cell lines.

The pSV40-5×HRE-luc-Kana reporter vector was constructed by subcloning the VspI-BamHI fragment of the pGL3-5×HRE-Control vector, bearing the SV40 promoter and the luciferase gene, into the VspI-BamHI site of the pEGFP-C2 vector. The pGL3-5×HRE-Control vector, containing five contiguous hypoxia responsive elements (5'-GATCTGAGACAGCACGTAGGGC-3') upstream of the luciferase reporter gene, was a generous gift from Dr. M. Geiszt (Institute of Physiology, Semmelweis University, Budapest, Hungary).

The SK-NF-I human neuroblastoma cell line and the CCF-STTG1 human astrocytoma cell line were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Normoxic cultures as well as samples treated with the iron chelator desferrioxamine (DFO; 100 μM final concentration) were kept in 21% O₂, 74% N₂ and 5% CO₂ in humidified atmosphere. Hypoxic samples were incubated in a humidified atmosphere of 1% O₂, 94% N₂ and 5% CO₂ in a modular incubator chamber (Billups-Rothenberg, USA). All reagents were of analytical grade and obtained from Sigma-Aldrich Co. Cell viability exceeded 95% throughout all experiments as proven by the trypan blue exclusion test.

In reporter assays, 1.5 × 10⁶ cells were transiently cotransfected with 0.3 μg pSV40-5×HRE-luc-Kana reporter plasmid and 0.1 μg pCMV-β-gal using the Lipofectamine reagent (Invitrogen). At 24 hrs after transfection the cells were subjected to hypoxia or incubated with DFO for 24 hrs, respectively, as indicated. Cells were extracted by three consecutive freeze-thaw cycles in Tris-HCl buffer (250 mM, pH 8.0), and luciferase and β-galactosidase activities were determined as reported earlier [50].

Cells were treated and washed as described above. Pellets were resuspended in a freshly prepared lysis buffer containing 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 10% (V/V) glycerol, 2 mM DTT, 0.5% (V/V) NP-40, 5 mM EDTA, 1 mM Na-vanadate, 1 mM PMSF, 20 mM NaF, 10 mM benzamidine, 10 mM lactacystin (a proteasome inhibitor), supplemented with Complete Protease Inhibitor Cocktail (Roche). Cells were disrupted by sonication on ice with a Vibra-Cell device (Sonics & Materials, USA) at 20 kHz and 25 W output by 3 × 10 s pulses. Supernatants were clarified by centrifugation (14,000 g, 20 min, 4°C). The protein concentration of cleared supernatants was determined with the Bio-Rad D_C Protein Assay kit. Samples were diluted to equal protein concentration and supplemented with equal volumes of 2× Laemmli buffer followed by heat denaturation (100°C, 5 min). Approximately 25 μg of total protein were resolved by SDS-PAGE on 8% gel slabs and subjected to western blotting as described in [51].

Membranes were immunoblotted with a polyclonal anti-human HIF-1α primary antibody at 1:2,000 dilution for 60 min and subsequently with a secondary anti-mouse antibody derived from goat at 1:4,000 dilution (60 min, room temperature). Immunocomplexes were visualized by the enhanced chemiluminescence reaction (Amersham Life Sciences). Three blots were made from 3 independent biological samples.

Total RNA was isolated by the RNeasy kit (Qiagen), according to the manufacturer's instructions. The quality of the preparation was checked by running an aliquot on ethidium bromide stained agarose gels.

cDNA was reverse transcribed with the High-Capacity cDNA Archive Kit (ABI). The reaction contained 0.2 μg total RNA, 5 U/μl MultiScribe™ Reverse Transcriptase and 1× relative concentration of Reverse Transcription Buffer, dNTPs and random primers in 50 μl final volume. The reaction was incubated at 25°C for 10 min and then at 37°C for 120 min.

Real-time PCR assays were performed in 25 μl final volume containing 5 μl cDNA, 1× ABI PCR master mix, gene-specific TaqMan^® primers and the gene-specific, FAM-labelled probe. Amplification and signal detection were performed using an ABI 7300 Real-Time PCR System (Applied Biosystems). Denaturation at 95°C, 10 min was followed by 40 thermocycles (95°C, 15 sec and 60°C, 1 min). Reactions were performed from three independent biological replicates in triplicate using RNase-free water as negative control. C_T-values were set in the exponential range of the amplification plots using the 7300 System Sequence Detection Software 1.3. ΔΔC_T-values corresponded to the difference between the C_T-values of the genes examined and those of the RPLP0 calibrator (internal control) gene. Relative expression levels of genes were calculated and expressed as 2^-ΔΔCT. To minimize the effect of pipetting errors, the TaqMan reaction mixture contained 6-carboxy-X-rhodamine (ROX) as a passive reference calibrator fluorescent dye.

The following TaqMan^® assays (Applied Biosystems) were used in this study: HGPRT (Hs99999909_m1); RPLP0 (Hs99999902_m1); HMBS (Hs00609297_m1); RPII (Hs00172187_m1); β-actin (Hs99999903_m1); VEGF (Hs00900058_m1); DRD2 (Hs01024460_m1); DRD3 (Hs00364455_m1); DRD4 (Hs00609526_m1); DRRF (Hs00259103_m1); COMT (Hs02511558_s1); MAOA (Hs00165140_m1); DAT (Hs00997371_m1).

2 × 10⁵ cells were grown on coverslips for 2 days in 1% oxygen. Then the cells were washed thrice with PBS and fixed for 20 min with 4% formalin. After fixation the coverslips were washed thrice with PBS, permeabilized with 0.1% Triton-X100, washed with PBS and blocked in PBS containing 5% fetal calf serum (FCS) for 1.5 hrs. α-DRD3 (sc-9114, Santa Cruz Biotechnology) and α-DRD4 (AB1787P, Millipore Co.) antibodies were diluted 1:50 and 1:250, respectively, in 1% FCS/PBS and applied to the cells overnight. After washing with FCS/PBS, the cells were incubated with diluted (1:1000), Alexa488-conjugated anti-rabbit secondary antibodies for 60 min, washed with PBS and mounted on glass slides with Mowiol (Polysciences). Fluorescence images were obtained and photographed in a Leitz Dialux 20 EB microscope equipped with epifluorescence optics. Staining intensities were quantitated by the ImageJ image processing software.

Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by the Tukey-Kramer Multiple Comparison Test (GraphPad InStat software).