Von Hippel-Lindau (VHL) tumor suppressor protein is expressed in neurons of the central nervous system and plays an important role during the neuronal differentiation of central nervous system progenitor cells. To elucidate the neuronal differentiating potential of VHL protein in neuroblastoma cells, we overexpressed or inhibited VHL protein in human neuroblastoma cells (SY-SH5Y), and examined the morphological change, expressions of neuronal markers, and electrophysiological functions. Here we show that with VHL gene transduction SY-SH5Y cells stably expressing the VHL protein had neurite-like processes with varicosities, showed the distinct expression of the neuronal markers neuropeptide Y and neurofilament 200, acquired regulated neurosecretion competence in response to depolarizing and cholinergic stimuli, and had large voltage-gated fast sodium currents and delayed rectifier potassium (Kv) currents compatible with those of functional neurons. In addition, they displayed inactivated ether-á-go-go potassium channels related to the promotion of the cell cycle and to the termination of differentiation. Also, by treatment with retinoic acid, they rapidly underwent cell death related to apoptosis. These findings suggest that the induction of neuronal function by VHL protein is associated with down-regulation of the cell cycle. In contrast, the inhibition of endogenous expression of VHL protein with antisense-orientated VHL gene transduction reduced such neuronal properties inherent to these cells, including the capacity for activation of ether-á-go-go channels. In conclusion, VHL protein has a neuronal differentiating potential to transform neuroblastoma cells into functional neuron-like cells. Our finding of the neuronal differentiation of neuroblastoma cells under the control of the VHL gene may contribute to the development of clinical techniques for neuronal regeneration in the case of intractable neuronal diseases and for differentiation therapy against neuroblastomas.

VHL4 tumor suppressor gene is the causative gene of VHL disease (1). Somatic mutations of the VHL gene are also often detected in sporadic cases of renal cell carcinoma (2) and CNS hemangioblastoma (3).

The human VHL gene encodes two VHL proteins, a protein of Mr 30,000 (pVHL30) consisting of the entire VHL open reading frame (amino acid residues 1–213; Ref. 4) and a protein of Mr 19,000 (pVHL19) arising as result of internal translation from the second methionine residue within the VHL open reading frame (residues 54–213; Ref. 5). pVHL19 contains a region adequate to function biologically (5) and a region highly conserved among humans, rats, and mice (1, 6).

pVHL mediates an important control mechanism for tumorigenesis and angiogenesis. Previous studies of the structure and function of pVHL revealed that pVHL activates the ubiquitin-mediated degradation pathway (7, 8) and negatively regulates the synthesis of HIF, which induces hypoxia-inducible genes such as VEGF (9). The pVHL β-domain has biochemical properties allowing inhibition of tumor growth and invasion (10), production of extracellular fibronectin matrix, and degradation of HIF-1α (11). β-Domain mutations inactivate VHL functions.

The VHL gene and pVHL are expressed specifically in the neuronal cells in the CNS (12, 13). We demonstrated recently that expression of pVHL was associated with neuronal differentiation of CNS progenitor cells. In addition, transduction of CNS progenitor cells with VHL gene led to rapid induction of neuronal differentiation, whereas inhibition of pVHL increased the cell-cycle activity and the nestin expression of CNS progenitor cells (14).

Neuroblastoma is a highly malignant pediatric tumor derived from the neural crest. Because neuroblastoma cells retain some characteristics of neural progenitor cells, they are able to undergo neuronal differentiation in the presence of appropriate agents. SH-SY5Y human neuroblastoma cells derived form sympathetic neuronal cells have often been used as one of the models for the analysis of neuronal function and differentiation (15). Many investigations have shown that when SH-SY5Y neuroblastoma cells are treated with RA, they undergo morphological, biochemical, and electrophysiological changes leading to neuronal differentiation (15, 16, 17, 18, 19, 20). Recently these cells were also searched for EAG potassium channels related to the promotion of cell cycles and the end of differentiation (21).

In previous studies tumor suppressor gene transduction alone without the presence of some bioactive agent such as RA has not transformed neuronal tumor cells such as SH-SY5Y cells into functional neuron-like cells having neuron-specific electrophysiological properties. This study sought to elucidate the neuronal differentiating potential of VHL protein for neuroblastoma cells on the basis of the morphological and functional changes in SH-SY5Y cells. Our results infer the possibility of clinical application of pVHL for neuronal intractable disease or differentiation therapy against neuroblastomas.

Vector Constructions.

VHLcDNA (g7-11; Ref. 22) was amplified by PCR using the following primers: 5′-CTGAATTCACCATGCCCCGGAGGGCGGAG-3′ and 5′-GAGAATTCTCAATCTCCCATCCGTTGATG-3′. The product contained cDNA spanning the entire human VHL open reading frame (residues 1–213, Mr 30,000). VHL cDNA (g7-11) was also PCR-amplified using the primers: 5′-CTGAATTCACCATGGAGGCCGGGCGGCCG-3′ and 5′-GAGAATTCTCAATCTCCCATCCGTTGATG-3′ to generate a product that contained VHL cDNA-encoding residues 54–213 (Mr 19,000). These products were subcloned into pcDNA 3.1 (Invitrogen, Carlsbad, CA) to generate pcDNA3-VHL30 (sense-oriented VHL30 expression plasmid), pcDNA3-VHL30R (antisense-oriented VHL30 gene plasmid), pcDNA3-VHL19 (sense-oriented VHL19 expression plasmid), and pcDNA3-VHL19R (antisense-oriented VHL19 gene plasmid). Moreover, a VHL β-domain mutant plasmid (pcDNA3-VHL-mut) was generated by deleting the domain encoding the 87 (Bgl1 site) -130 (Hpa1 site) amino acid sequence within the full-length VHL cDNA. The β-domain mutants lacking this domain lose VHL functions, as was mentioned in “Introduction” (10, 11). VHL cDNA sequences of these plasmids were confirmed with a DNA autosequencer (ABI Prism 310 Genetic Analyzer; Perkin-Elmer, Foster City, CA).

Cell Culture and Transfections.

SH-SY5Y human neuroblastoma cells (wild-type cells), kindly provided by Dr. Nakagawara, Chiba Prefectural Cancer Institute, Chiba, Japan, were maintained in DMEM and 10% FBS. These cells were transfected with pcDNA3.1, pcDNA3-VHL30, pcDNA3-VHL30R, pcDNA3-VHL19, pcDNA3-VHL19R, or pcDNA3-VHL-mut with Effectene Transfection Reagent (Qiagen, Valencia, CA). Transfected cells were selected with G418 (480 μg/ml; Genecitin; Life Technologies, Inc.) to establish stable transfectants designated as empty cells, VHL30 cells, VHL30-R cells, VHL19 cells, VHL19-R cells, and VHL-mut cells, respectively. Established stable clones were maintained in DMEM with 10% FBS and 200 μg/ml of G418.

Microscopic Examination and Cell Counting.

Cultured cells were observed by phase-contrast microscopy. The percentage of cells bearing neurite-like processes with varicosities was calculated based on a count of 1000 cells.

Treatment with RA for Stable VHL-expressing and VHL-inhibited Cells.

Cells were seeded at 2 × 104 cells in six-well plates, and 2 days later RA was added for a final concentration of 10−6m to the culture medium. Viable cells were counted by the trypan blue exclusion method at various times, and morphological changes in the cultured cells were observed with a phase-contrast microscope. Moreover, cultured cells were fixed in 1% paraformaldehyde at 24, 48, and 72 h after the addition of the RA; and apoptosis was detected by using the TUNEL assay (Trevigen, Gaithersburg, MD) according to the manufacturer’s instructions. The quantification of apoptotic cells was done by calculating as the percentage of the total cells evaluated that were TUNEL-positive (>1000 cells/sample).

Semiquantitative RT-PCR Analysis.

Total RNA was extracted from SY5Y cells by the guanidinium isothiocyanate/acid phenol method using TRIzol RNA extraction reagent (Life Technologies, Inc.) and was reverse-transcribed by using an oligodeoxythymidylic acid primer and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). The synthesized cDNAs were amplified by PCR primer sets and PCR conditions as described previously about NPY (23) and β-actin (24). The PCR cycles (26 and 22 cycles for NPY and β-actin, respectively) were determined beforehand to be in the linear range before the plateau effect with PCR amplification. PCR products were analyzed quantitatively with the NIH image software program, and the NPY mRNA level was normalized to that of β-actin mRNA.

Antibodies.

To generate affinity-purified rabbit polyclonal VHL antibody pAbVHLGST, rabbits were immunized with a bacterially produced GST-human pVHL (residues 1–213) fusion protein, and the polyclonal sera were affinity-purified with a GST-pVHL column as described previously (4). Other antibodies used in this study were mouse monoclonal anti-VHL antibodies [mAbVHL06 (Ref. 25), kindly provided by Dr. Sakashita, Kumamoto University, Japan, and IG32 from PharMingen, San Diego, CA], mouse monoclonal antineurofilament 200 antibody (N52; Sigma, St. Louis, MO), rabbit antineurofilament 200 IgG fraction of antiserum (Sigma), rabbit polyclonal NPY antibody (Peninsula Inc., San Carlos, CA), and antiactin polyclonal antibody (BT560; Biomedical Technologies Inc., Stoughton, MA).

Cell Lysis, Immunoprecipitation, and Western Blot Analysis.

Cultured cells (5 × 106) were washed with ice-cold PBS and lysed in Triton X-100 lysis buffer (26). The total protein concentration of each lysate was adjusted to 2 mg/ml, and lysates were immunoprecipitated with mAbVHL06. The immunoprecipitates were analyzed by SDS/15% PAGE and Western blotting with pAbVHLGST (1:500 dilution). For immunoblotting of NFH and actin, lysates were electrophoresed on SDS/5% and SDS/10% polyacrylamide gels, respectively; and Western blots were probed with rabbit anti-NFH IgG (1:500 dilution) or BT560 (1:500 dilution). Immunolabeled bands were detected by using enhanced chemiluminescence reagents (Amersham Pharmacia).

Immunocytochemistry.

Cultured cells harvested in Lab-Tek chamber slides (Nalge Nunc, Rochester, NY) were fixed with cold-acetone:methanol (1:1) for 20 min at 4°C, and stained with IG32 (1:200 dilution) and NPY antibody (1:200 dilution) for detection of pVHL and NPY, respectively, or with pAbVHLGST (1:200) and N52 (1:200) for pVHL and NFH, respectively. Cells were then reacted with fluorescein isothiocyanate-labeled donkey antirabbit IgG (1:100; Amersham Pharmacia) or biotinylated horse antimouse IgG (1:100; Vector Laboratories Inc., Burlingame, CA) and Texas Red-avidine (1:100; Amersham Pharmacia). Double-stained cells were observed with a laser scanning confocal microscope (Bio-Rad MRC1000 laser confocal system; Bio-Rad Laboratories, Hercules, CA).

ELISA.

The concentration of NPY in conditioned medium was analyzed by ELISA (NPY Enzyme Immunoassay kit; Peninsula Inc.) according to the manufacturer’s instructions. Cells were seeded at 8 × 105 cells in six-well plates, and the medium was then changed to fresh medium (1% FBS in DMEM) 6 h later and harvested after another 24 h. To evaluate regulated secretion of NPY, we added the depolarizing stimulant KCl or the cholinergic muscarinic agonist carbachol to each medium at a final concentration of 100 mm or 1 mm, respectively, and stimulated the cells with these agents for 5 min in the presence or absence of EGTA (2 mm), a chelator of extracellular calcium. The NPY concentrations were calculated from the standard curve of synthesized NPY (Peninsula Inc.).

Electrophysiology.

Whole-cell voltage-clumped recordings were made of cells standing alone to ensure sufficient voltage and space clamp control.

To record fast sodium and delayed rectifier potassium (Kv) currents, we prepared the extracellular and intracellular solutions as described previously (18). A holding potential of −80 mV and voltage step of 10 mV over the range of −100 to 100 mV with 20-msec durations were applied to the recorded cells through the patch electrodes.

For recording EAG channel currents, the extracellular and intracellular solutions were prepared as described previously (21). A holding potential of -100 mV and voltage step of 10 mV over the range of −60 to +80 mV with 1000-msec durations were applied for the recording of EAG currents.

For recordings and data analysis we used CEZ-2300 (Nihon Kohden, Tokyo, Japan) and pCLAMP 6.0 software (Axon Instruments, Burlingame, CA). Linear components of leak and capacitive currents were reduced by analogue circuitry and then canceled by the P/N method. Signals were sampled every 20 μsec, and currents were filtered at 5 kHz. Data were additionally processed with Origin 5.0 (Microcal, Northhampton, MA).

Establishment of Stable VHL-expressing and VHL-inhibited Cells.

Wild-type cells and cells transfected with empty vector (empty cells) showed endogenous expression of only pVHL19 by Western blot analysis (Fig. 1,A). Cells stably expressing pVHL19 or pVHL30 at high levels were isolated as VHL19 cells (V06-1 and V06-3) or VHL30 cells (V02-1 and V02-4), respectively (Fig. 1,A). VHL30 cells also expressed endogenous pVHL19 as well as exogenous pVHL30 (Fig. 1 A), which showed multiple bands in the vicinity of Mr 30,000, as demonstrated previously (27).

Cells with inhibited expression of pVHL were isolated as VHL-inhibited cells (R01-4 and R13-1) from cells transfected with pcDNA3-VHL19R (VHL19-R cells; Fig. 1 A) and not those transfected with pcDNA3-VHL30R (VHL30-R cells). VHL30-R cells were used as a nonrelevant antisense control.

pVHL Induces Morphological Neuronal Changes.

Treatment of SH-SY5Y cells with phorbol esters and staurosporine induces distinct morphological changes toward neurons including the formation of neurite-like processes with abundant varicosities, which reflect the functional properties of nerve terminals (28). Neurite-like processes with varicosities were observed by phase-contrast microscopy (Fig. 1,B). To discriminate neurites from nonspecific stress fibers, we excluded cells bearing only processes without varicosities from the total number of cells showing this morphological differentiation. VHL19 cells showed morphological changes in 16–21% of counted cells and VHL30 cells, in 8–11% (Fig. 1 C). However, few other cell groups showed such morphological changes.

pVHL Induces Rapid Cell Death Related to Apoptosis with RA.

VHL-expressing cells proliferated significantly slower than other groups before treatment with RA (Fig. 2,A). The treatment with RA induced the death of ∼90% of the VHL-expressing cells by 3 days, whereas VHL-inhibited cells, wild-type ones, and empty cells were not induced to undergo rapid cell death, but showed decreased cell proliferation (Fig. 2,A) and neurite extension. Most of the VHL-expressing cells induced to rapid cell death by RA were TUNEL-positive, i.e., apoptotic; and their number of apoptotic cells was significantly greater than that for the other cell groups (Fig. 2 B).

Expression of Neuronal Markers, NPY, and Neurofilament 200 Depends on That of pVHL.

NPY is a neurotransmitter and neuromodulator, and the induction of neuronal differentiation is associated with the persistence of NPY in neuroblastoma cell lines including SH-SY5Y (16).

NPY mRNA expression was detected at much higher levels in VHL-expressing cells than in wild-type and empty controls by semiquantitative RT-PCR (Fig. 3 A). In contrast, VHL-inhibited cells expressed NPY mRNA at very low levels.

VHL-expressing cells, especially VHL19 cells, showed many large fluorescent speckles of NPY uniformly distributed in their cytoplasm by immunofluorescence imaging (Fig. 3 C). These speckles were composed of aggregates of NPY punctates including varicosities. Wild-type, empty, antisense control (VHL30-R cells; data not shown), and VHL-mut cells showed ubiquitous NPY expression in cell bodies with only a few small NPY speckles. In contrast, VHL-inhibited cells showed weak heterogeneous NPY staining of cell bodies without speckles.

NFH is the largest subunit of triplet neurofilaments and an essential component of the neurofilaments synthesized by mature neurons (29). NFH is heavily phosphorylated, and pNFH is more extensively present in axons than is the nonphosphorylated one; and dpNFH is the form required for cytoplasmic interactions in neurons (30). NFH is usually little expressed in wild-type SH-SY5Y cells (17).

pNFH was weakly expressed in wild-type and empty cells, and more highly expressed in VHL-expressing cells by Western blot analysis (Fig. 3,B), especially in the VHL19 cells. VHL19 cells and VHL30 cells also expressed dpNFH. In contrast, VHL-inhibited cells expressed no NFH at all. Actin, non-neuronal structural protein, was almost equally expressed in all of the groups (Fig. 3 B).

Both VHL19 and VHL30 cells definitely expressed NFH, as judged from the results of double-staining immunocytochemistry for the detection of pVHL and NFH; and it was colocalized with pVHL (Fig. 3 D). Wild-type, empty, antisense control (data not shown), and VHL-mut cells expressed NFH very weakly; and VHL-inhibited cells expressed very little NFH.

pVHL with a mutated β-domain was detected by pAbVHLGST, a polyclonal anti-VHL antibody (Fig. 3,D), but not by IG32, a monoclonal one (Fig. 3 C). The overexpression of this mutated pVHL was also confirmed by Western blot with pAbVHLGST, which showed immunolabeled bands in the vicinity of Mr 23,000 (data not shown).

pVHL Affords Regulated Neurosecretion Competence to SH-SY5Y Cells.

To evaluate regulated neurosecretion, we measured the concentration of NPY, a neurotransmitter, in the conditioned medium by using ELISA (Fig. 4). Wild-type controls, empty controls, and VHL-inhibited cells showed no significant difference from one another in their basal concentration. In contrast, the conditioned medium from VHL-expressing cultures had significantly higher concentrations than that from any of the other groups. Depolarizing stimulation by the addition of KCl did not significantly increase the NPY concentration compared with the basal condition in wild-type controls, empty controls, or VHL-inhibited cells. In contrast, the NPY concentration increased 1.9–2.4-fold compared with the basal condition in VHL-expressing cells. Cholinergic stimulation with carbachol caused a significant increase in NPY release only in VHL-expressing cells, which resulted in a level 1.6–2.0-fold higher than the basal one. KCl and carbachol-evoked NPY secretion was suppressed by blocking extracellular calcium with its chelator, EGTA, demonstrating the calcium-dependent nature of the process.

pVHL Affects Voltage-gated Na+ Channels and Delayed Rectifier K+ (Kv) Ones.

To evaluate functional expression of voltage-gated ion channels, we assessed the voltage-gated fast sodium currents and/or fast delayed rectifier potassium ones by patch-clamping and recording in a voltage-clamped configuration, recorded over a short duration up to 20 ms (Fig. 5, A and B).

Voltage-activated fast sodium currents, which are the initial inward currents and form the ionic basis of the neuronal action potential, showed maximum currents of 603 ± 20 pA, 616 ± 36 pA, and 619 ± 21 pA for wild-type controls, empty controls, and VHL-inhibited cells, respectively. In contrast, VHL-expressing cells, especially the VHL19 cells, showed significantly higher currents of 1477 ± 211 pA and 1116 ± 79 pA for the VHL19 cells and VHL30 cells, respectively. These voltage-activated sodium currents were completely suppressed by addition of 0.5 μm tetrodotoxin (Fig. 6 A, bottom panel).

Voltage-gated fast delayed rectifier potassium currents, which have been shown recently to originate from Kv channels (19), showed maximum values of 504 ± 5.3 pA, 538 ± 65 pA, and 118 ± 16 pA for wild-type controls, empty controls, and VHL-inhibited cells, respectively. Although there were no significant differences in the currents between wild-type and empty controls, the currents of the VHL-inhibited cells were significantly lower than those of the wild-type or empty controls. In contrast, maximum currents of VHL-expressing cells were 1624 ± 389 pA and 1121 ± 67 pA for the VHL19 cells and VHL30 cells, respectively, which were 2–3-fold higher than those of the wild-type or empty controls.

pVHL Down-Regulates EAG Potassium Channels.

EAG potassium channel currents are involved in the cell cycle (31), tumor progression (32) and the termination of differentiation (21). Marked down-regulation of EAG was earlier observed in RA-induced neuronal differentiation of SH-SY5Y cells (21). So we investigated whether or not pVHL could influence the EAG channels.

EAG channel currents were detected in 54% and 50% of recorded cells in wild-type and empty controls, respectively (Fig. 6,B). Maximum currents of cells bearing EAG channels were 401 ± 37 pA and 388 ± 40 pA, respectively (Fig. 6,B). There were no significant differences between the groups. EAG currents of VHL-inhibited cells were detected in 77% of the recorded cells, which was significantly more than in wild-type and empty controls. The maximum current of cells bearing EAG channels was 1478 ± 125 pA, which was much larger than in the controls. In contrast, neither VHL19 cells nor VHL30 cells showed EAG channel currents. Instead, most VHL-expressing cells showed fast delayed rectifier potassium (Kv) currents (Fig. 6 A).

This study demonstrated the relationship between pVHL and the induction of neuronal function in SH-SY5Y cells by both overexpression and inhibition of pVHL. VHL-expressing cells, especially VHL19 cells, showed neurite-like processes with varicosities, which indicate the morphological phenotype of mature neurons. These cells also distinctly expressed the neuronal structural protein NFH and the neurotransmitter NPY. Expression of NFH was colocalized with that of pVHL. These cells also demonstrated potassium- and carbachol-evoked NPY release into the culture medium, as well as increased basal NPY release. This result suggests that VHL-expressing cells had acquired regulated neurosecretion competence. This neurosecretion response was a calcium-dependent one, which indicates calcium influx in response to depolarizing stimuli at neuronal voltage-gated calcium channels (33). When whole-cell currents were recorded under voltage-clamp conditions, VHL-expressing cells had large voltage-gated fast sodium currents and large fast delayed rectifier potassium (Kv) currents compatible with those of mature neurons. In addition, none of the VHL-expressing cells showed EAG channel currents. This result suggests that pVHL down-regulates EAG channels, which have been associated with cell proliferation and the termination of differentiation (21). In all of the experiments in this study, VHL19 cells showed the most influential effects related to neuronal differentiation. pVHL19 is known to have bioactivity in the induction of neuronal function as well as in other functions, as demonstrated previously (5).

In contrast, VHL-inhibited cells showed decreased expression of NPY and NFH, and significantly lower potassium channel currents compared with wild-type and empty controls. Moreover, EAG currents were larger in many VHL-inhibited cells compared with those in the controls. Inhibition of pVHL would thus seem to activate EAG channels and cell growth.

Cells overexpressing pVHL with a mutated β-domain did not show morphological neuronal changes and did not influence expression of NPY and NFH. These results suggest this domain to be indispensable for the induction of neuronal function.

These findings show that pVHL is essential for neuronal properties inherent in human neuroblastoma cells and suggest that this protein has a unique potential to cause the differentiation resulting in functional neuron-like cells morphologically, biochemically, and physiologically.

The relationship between the VHL gene and cellular differentiation is supported by previous findings as well. VHL expression morphologically induced the cellular differentiation of renal cell carcinoma cells into the epithelial phenotype of renal tubular cells (34). We found recently that the VHL gene was involved in the neuronal differentiation of rodent neural progenitor cells (14). Basic helix-loop-helix transcription factors including MASH-1 (35) and NeuroD (36), as well as SOX-1 (37), a HMG-box protein related to SRY, were found to be the independent inducers of neuronal function and the determination factors of neural fate. Here we showed similarly that pVHL made a critical contribution to neuronal differentiation in SH-SY5Y cells and allowed these cells to function as mature neuron-like cells.

The mechanisms by which pVHL induces neuronal function are yet unknown, but there are some possibilities in the light of recent studies. First, pVHL can interact with protein kinase C, which is related to neuronal differentiation (38). Second, pVHL regulates VEGF165 through HIF-1. VEGF165 functionally competes with semaphorin 3, a mediator of neuronal cell guidance, for the same receptor, neuropillin 1 (9, 39, 40). Third, VHL-mediated down-regulation of the cell cycle (41) may be associated with the induction of neuronal function. In CNS progenitor cells the expression of pVHL is associated with neuronal differentiation as well as with down-regulation of the cell cycle, whereas inhibition of pVHL with VHL mRNA antisense oligonucleotide promoted the cell cycle (14). Our present data show that VHL-expressing cells proliferated much more slowly than other cell groups, and were induced to undergo rapid cell death related to apoptosis in the presence of RA. Moreover these cells did not show EAG currents, the disappearance of which suggests G2-M exit from the cell cycle (21).

Neuronal cell lines including neuroblastoma cells have been explored as transplantation materials for the treatment of intractable neurological diseases such as Parkinson’s disease, spinal cord injury, and cerebral infarction. Transplantation of neuroblastoma cells improved the neurological deficit seen in a Parkinson’s rat model (42, 43). Neuronal cells (NT2N cells) derived from an embryonal carcinoma cell line by treatment of RA promoted functional recovery of ischemia and spinal cord injury (44, 45). Moreover, transplantation of immortalized neural progenitor cells led to resolution of stroke deficit (46). pVHL-expressing SH-SY5Y neuroblastoma cells and pVHL-overexpressing rodent neural progenitor cells markedly improved rotational behavior in 6-hydroxydopamine-lesioned Parkinson model rats (preliminary data). VHL-expressing neural cells including neuroblastoma cells could form neuronal networks in the brain and function as dopamine-secreting neurons. They may be used in clinical applications as transplantation material for the treatment of intractable neurological diseases such as those mentioned above.

In addition, the induction of neuronal function in neuroblastoma cells under the control of the VHL gene would make a contribution to differentiation therapy against neuroblastoma. RA induces growth arrest and differentiation of neuroblastoma cells; and clinical trials of retinoid treatment for neuroblastoma showed increased survival of neuroblastoma patients (47). IFN-γ or herbimycin combined with RA increased the induction of neuronal differentiation in neuroblastoma cells (20). Our data indicate that pVHL-overexpressing cells rapidly died via an apoptosis in the presence of RA. VHL gene transduction therapy for neuroblastoma patients should be effective and be even more powerful in combination with RA.

Fig. 1.

A, Western blot analysis of pVHL. Stable VHL-expressing cells (VHL19 and VHL30) and VHL-inhibited cells (VHL-R) were established from ∼50 colonies of each. The bands close to Mr 30,000 (∗) represent mouse IgG (mAbVHL06) used in immunoprecipitation. WT, wild-type cells; empty, empty control cells. B, phase-contrast photomicrograph of VHL-expressing cells (V06–1). The cells show neurite-like processes with varicosities. Arrowheads point to the varicosities. Scale bar, 15 μm. C, percentage of cells bearing neurite-like processes with varicosities. ∗∗P < 0.001, one-way ANOVA. Data are the mean from three independent visual fields; bars, ±SE. ASC, antisense control cells (VHL30-R cells).

Fig. 1.

A, Western blot analysis of pVHL. Stable VHL-expressing cells (VHL19 and VHL30) and VHL-inhibited cells (VHL-R) were established from ∼50 colonies of each. The bands close to Mr 30,000 (∗) represent mouse IgG (mAbVHL06) used in immunoprecipitation. WT, wild-type cells; empty, empty control cells. B, phase-contrast photomicrograph of VHL-expressing cells (V06–1). The cells show neurite-like processes with varicosities. Arrowheads point to the varicosities. Scale bar, 15 μm. C, percentage of cells bearing neurite-like processes with varicosities. ∗∗P < 0.001, one-way ANOVA. Data are the mean from three independent visual fields; bars, ±SE. ASC, antisense control cells (VHL30-R cells).

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Fig. 2.

A, cell growth and the influence of treatment with RA are shown. Two days after the cells were seeded, RA was added to the culture medium (arrowhead). Viable cells was measured by trypan blue exclusion for 0–7 days. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, one-way ANOVA. WT, wild-type cells; empty, empty control cells; VHL-R, VHL-inhibited cells; VHL19, pVHL19-expressing cells; VHL30, pVHL30-expressing cell. B, apoptosis was evaluated by the TUNEL assay at 0, 24, 48, 72 h after the addition of RA. Percentage of apoptotic cells was quantitated from three independent experiments. ∗P < 0.0005, ∗∗P < 0.0001 compared with wild-type or empty controls, Student’s t test. Data are presented as the means; bars, ±SE.

Fig. 2.

A, cell growth and the influence of treatment with RA are shown. Two days after the cells were seeded, RA was added to the culture medium (arrowhead). Viable cells was measured by trypan blue exclusion for 0–7 days. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, one-way ANOVA. WT, wild-type cells; empty, empty control cells; VHL-R, VHL-inhibited cells; VHL19, pVHL19-expressing cells; VHL30, pVHL30-expressing cell. B, apoptosis was evaluated by the TUNEL assay at 0, 24, 48, 72 h after the addition of RA. Percentage of apoptotic cells was quantitated from three independent experiments. ∗P < 0.0005, ∗∗P < 0.0001 compared with wild-type or empty controls, Student’s t test. Data are presented as the means; bars, ±SE.

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Fig. 3.

A, semiquantitative RT-PCR of mRNAs for NPY and β-actin. The intensity of NPY mRNA expression is shown as the ratio to β-actin. ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05, one-way ANOVA. Data are the mean ratio from 3 independent experiments; bars, ±SE. WT, wild-type cells; empty, empty control cells; VHL-R, VHL-inhibited cells; VHL19, pVHL19-expressing cells; VHL30, pVHL30-expressing cells. B, Western blot analysis of NFH and actin. Human normal brain homogenates are shown as the positive control for pNFH and dpNFH. C, immunofluorescence double-staining analysis of pVHL and NPY. Photos show a sample from each group. Scale bar, 50 μm. The insets have been magnified ×10. VHL-mut, VHL-mut cells. D, immunofluorescence double-staining analysis of pVHL and NFH. Scale bar, 50 μm. The insets have been magnified ×10.

Fig. 3.

A, semiquantitative RT-PCR of mRNAs for NPY and β-actin. The intensity of NPY mRNA expression is shown as the ratio to β-actin. ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05, one-way ANOVA. Data are the mean ratio from 3 independent experiments; bars, ±SE. WT, wild-type cells; empty, empty control cells; VHL-R, VHL-inhibited cells; VHL19, pVHL19-expressing cells; VHL30, pVHL30-expressing cells. B, Western blot analysis of NFH and actin. Human normal brain homogenates are shown as the positive control for pNFH and dpNFH. C, immunofluorescence double-staining analysis of pVHL and NPY. Photos show a sample from each group. Scale bar, 50 μm. The insets have been magnified ×10. VHL-mut, VHL-mut cells. D, immunofluorescence double-staining analysis of pVHL and NFH. Scale bar, 50 μm. The insets have been magnified ×10.

Close modal
Fig. 4.

Secretion of NPY was evaluated by ELISA. The NPY concentration was measured under the following conditions: basal, stimulation with KCl or carbachol, and the addition of EGTA with either stimulant. ∗P < 0.001 compared with wild-type or empty cells; #P < 0.01; ##P < 0.005 compared with basal concentration; ¶P < 0.05; ¶¶P < 0.01 compared with potassium or carbachol-evoked secretion; Student’s t test. Data are presented as the mean concentration from three independent experiments; bars, ±SE. WT, wild-type cells; empty, empty control cells; VHL-R, VHL-inhibited cells; VHL19, pVHL19-expressing cells; VHL30, pVHL30-expressing cells.

Fig. 4.

Secretion of NPY was evaluated by ELISA. The NPY concentration was measured under the following conditions: basal, stimulation with KCl or carbachol, and the addition of EGTA with either stimulant. ∗P < 0.001 compared with wild-type or empty cells; #P < 0.01; ##P < 0.005 compared with basal concentration; ¶P < 0.05; ¶¶P < 0.01 compared with potassium or carbachol-evoked secretion; Student’s t test. Data are presented as the mean concentration from three independent experiments; bars, ±SE. WT, wild-type cells; empty, empty control cells; VHL-R, VHL-inhibited cells; VHL19, pVHL19-expressing cells; VHL30, pVHL30-expressing cells.

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Fig. 5.

A, electrophysiological analysis. Voltage-gated inward and outward currents were recorded in the whole-cell patch-clamp configuration. Inward and outward currents represent fast sodium currents and fast delayed rectifier potassium currents, respectively. Here the currents of empty controls and VHL19 cells are shown as typical cases. B, maximum sodium currents were measured as the peak of inward currents. Maximum fast delayed rectifier potassium currents were measured at 15 ms after voltage step onset. ∗P < 0.01, ∗∗P < 0.001 compared with sodium currents of wild-type or empty controls; #P < 0.01, ##P < 0.001 compared with potassium currents of each control; Student’s t test. Data represent the mean maximum currents (n = 6); bars, ±SE. WT, wild-type cells; empty, empty control cells; VHL-R, VHL-inhibited cells; VHL19, pVHL19-expressing cells; VHL30, pVHL30-expressing cells.

Fig. 5.

A, electrophysiological analysis. Voltage-gated inward and outward currents were recorded in the whole-cell patch-clamp configuration. Inward and outward currents represent fast sodium currents and fast delayed rectifier potassium currents, respectively. Here the currents of empty controls and VHL19 cells are shown as typical cases. B, maximum sodium currents were measured as the peak of inward currents. Maximum fast delayed rectifier potassium currents were measured at 15 ms after voltage step onset. ∗P < 0.01, ∗∗P < 0.001 compared with sodium currents of wild-type or empty controls; #P < 0.01, ##P < 0.001 compared with potassium currents of each control; Student’s t test. Data represent the mean maximum currents (n = 6); bars, ±SE. WT, wild-type cells; empty, empty control cells; VHL-R, VHL-inhibited cells; VHL19, pVHL19-expressing cells; VHL30, pVHL30-expressing cells.

Close modal
Fig. 6.

A, EAG currents of VHL-inhibited cells (VHL-R) and empty control cells (empty) are shown (top and middle panels, respectively). In contrast, none of the VHL-expressing cells showed EAG currents. Instead, fast delayed rectifier potassium currents were detected in most of them (bottom panel). B, percentage of cells bearing EAG channel currents is shown on the left vertical axis. None of the VHL-expressing cells (VHL19 and VHL30) showed EAG currents. ∗P < 0.01, ∗∗∗P < 0.0001 compared with sodium currents of wild-type (WT) or empty control cells (empty); Fisher’s extract probability test. The intensity of EAG currents of cells bearing EAG channels is shown on the right vertical axis. VHL-expressing cells were not measured because they showed no EAG currents. ##P < 0.001 compared with each control, Student’s t test. Data represents the mean maximum currents; bars, ±SE.

Fig. 6.

A, EAG currents of VHL-inhibited cells (VHL-R) and empty control cells (empty) are shown (top and middle panels, respectively). In contrast, none of the VHL-expressing cells showed EAG currents. Instead, fast delayed rectifier potassium currents were detected in most of them (bottom panel). B, percentage of cells bearing EAG channel currents is shown on the left vertical axis. None of the VHL-expressing cells (VHL19 and VHL30) showed EAG currents. ∗P < 0.01, ∗∗∗P < 0.0001 compared with sodium currents of wild-type (WT) or empty control cells (empty); Fisher’s extract probability test. The intensity of EAG currents of cells bearing EAG channels is shown on the right vertical axis. VHL-expressing cells were not measured because they showed no EAG currents. ##P < 0.001 compared with each control, Student’s t test. Data represents the mean maximum currents; bars, ±SE.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Nos. 11877244 and 13557120), and Yokohama Medical Research Foundation.

4

The abbreviations used are: VHL, Von Hippel-Lindau; CNS, central nervous system; pVHL, Von Hippel-Lindau gene product; HIF, hypoxia-inducible factor; RA, retinoic acid; EAG, ether-á-go-go; FBS, fetal bovine serum; VHL-mut, Von Hippel-Lindau-mutant; TUNEL, terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling; RT-PCR, reverse transcription-PCR; NPY, neuropeptide Y; GST, glutathione S-transferase; NFH, neurofilament-200; pNFH, phosphorylated neurofilament-200; dpNFH, dephosphorylated neurofilament-200; VEGF, vascular endothelial growth factor.

We thank Professor Masahiko Hosaka (Yokohama City University School of Medicine) for critical discussions, and also thank Teruyo Watanabe for technical assistance.

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