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Keywords:

  • Major vault protein;
  • Multidrug resistance;
  • Epilepsy;
  • Ganglioglioma;
  • Neurons;
  • Astrocytes

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Summary: Purpose: Recent evidence has been obtained that the major vault protein (MVP) may play a role in multidrug resistance (MDR). We investigated the expression and cellular localization of MVP in gangliogliomas (GGs), which are increasingly recognized causes of chronic pharmacoresistant epilepsy.

Methods: Surgical tumor specimens (n = 30), as well as peritumoral and control brain tissues, were examined for the cellular distribution pattern of MVP with immunocytochemistry. Western blot analysis showed a consistent increase in MVP expression in GGs compared with that in control cortex.

Results: In normal brain, MVP expression was below detection in glial and neuronal cells, and only low immunoreactivity (IR) levels were detected in blood vessels. MVP expression was observed in the neuronal component of 30 of 30 GGs and in a population of tumor glial cells. In the majority of the tumors, strong MVP IR was found in lesional vessels. Perilesional regions did not show increased staining in vessels or in neuronal and glial cells compared with normal cortex. However, expression of MVP was detected in the hippocampus in cases with dual pathology.

Conclusions: The increased expression of MVP in GGs is another example of an MDR-related protein that is upregulated in patients with refractory epilepsy. Further research is necessary to investigate whether it could play role in the mechanisms underlying drug resistance in chronic human epilepsy.

Gangliogliomas (GGs) are the most common tumor type in young patients with chronic focal intractable epilepsy (1–3). Although they may occur throughout the central nervous system, the temporal lobe is the most common location. GGs consist of a mixture of glial and neuronal elements. This histologic composition, which also is a prominent feature of glioneuronal hamartias, has attracted considerable interest with respect to the origin, as well as the high epileptogenicity of these lesions (2,3,4–6). A maldevelopmental nature has been proposed for this tumor entity (3,7–9).

Resistance to pharmacologic treatment with a broad range of antiepileptic drugs (AEDs) is another characteristic of these lesions (2,10–12). The basis of this multidrug resistance (MDR) is still elusive, but is likely to be multifactorial, involving several nonspecific mechanisms responsible for different types of clinical drug resistance, for example, as seen with drug resistance to cytostatic drugs in cancer treatment. One possible mechanism to account for a broad medical intractability, involving AEDs with different actions, is inadequate drug concentration in the epileptogenic areas. In recent years, attention has been focused on multidrug transporters, such as P-glycoprotein (P-gp) and the family of MDR-associated proteins (MRPs) [reviewed in (13–15)]. We recently showed overexpression of these multidrug transporters involving glial, neuronal, and endothelial cells in surgical resection specimens from patients with both focal cortical dysplasia (FCD) and GG (16,17). However, other mechanisms, such as intracellular transport and drug sequestration into exocytotic vesicles, also may play a role in clinical drug resistance. Evidence points to subcellular particles called vaults for their role in such a mechanism (18,19).

Vaults are multimeric RNA–protein complexes, with one predominant structural component, the major vault protein (MVP). The high evolutionary conservation (20) and broad distribution of vaults (21) suggest a basic physiologic cellular function. The detection of high expression levels in tissues potentially exposed to toxins and in macrophages supports the notion that the physiologic function of these molecules is to provide protection (21,22). Vaults are localized mainly in the cytoplasm, but a small fraction also has been localized at the nuclear membrane and the nuclear pore complex (23). Thus, although vault function remains undetermined, evidence supports the role of vaults in the vesicular transport of several compounds, mediating a bidirectional nucleocytoplasmic exchange (23–27).

The functional role of MVP has increased in significance in view of the finding the MVP vaults are overexpressed in many human tumors, and a connection has been shown between their expression and MDR (21,22,26,28–32). Although a direct involvement of vaults in MDR could not be demonstrated in the MVP knockout mouse model (33), it cannot be excluded that other mechanisms of drug resistance become upregulated on disruption of MVP and that vaults might still be involved in the protection against long-term exposure to drugs. In addition, recent evidence supports a role of vaults in other cellular process, such as cellular differentiation (34).

In the present study, Western blot and immunocytochemistry with antibodies (Abs) specific for MVP were performed in surgical specimens of patients with GGs and pharmacoresistant epilepsy. Our major aim was to provide data that may help to define the expression level and cellular distribution of MVP in tumor, peritumoral, and normal brain tissue, and may provide better insights into the mechanisms underlying MDR to treatment with AEDs in patients with chronic focal epilepsy.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Subjects

The 30 cases included in this study were obtained from the files of the departments of neuropathology of the Academical Medical Center (University of Amsterdam) and the University Medical Center in Utrecht. Patients underwent resection of GGs for medically intractable epilepsy. Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. Two neuropathologists reviewed all cases independently, and confirmed the diagnosis of GGs according to the revised World Health Organization (WHO) classification of tumors of the nervous system (35). From this material we selected six cases that contained sufficient amounts of perilesional zone (normal-appearing cortex/white matter adjacent to the lesion), for comparison with the normal-appearing control cortex/white matter and hippocampus (autopsy specimens from six age-matched patients without history of seizures or other neurologic diseases). Normal tissue adjacent to the lesional zone represents good-disease-control tissue, because tissue parameters such as seizure activity, drug exposure, fixation time, age, and sex are the same. Comparison with tissue from patients without pharmacoresistant epilepsy was not possible. This tissue was not available (patients responsive to AEDs do not normally undergo surgical resection for epilepsy).

Although the presence of cortical dysplasia was previously reported in GGs of our series (2), the cases selected for this study did not include cortical malformations. Histologically nonsclerotic hippocampus (non-HS; n = 3) and sclerotic hippocampus (HS, with cell loss involving CA1, CA3 sectors; n = 5) from GG patients undergoing extensive surgical resection of the mesial structures for the treatment of medically intractable complex partial epilepsy also was analyzed to test the effect of recurrent seizures, AEDs, and gliosis on the expression of MVP. Table 1 summarizes the clinical features (derived from patients' medical records) with particular attention to the characteristics of seizures (type and frequency of seizures, age at seizure onset). In all patients, the lesion was localized by brain magnetic resonance imaging (MRI) and computed tomography (CT) scan. Video-monitoring and intraoperative electrocorticography also were part of the presurgical evaluation. The predominant type of seizure pattern was that of complex partial seizures, which were resistant to maximal doses of AEDs. Pharmacoresistant epilepsy was defined by the presence of repetitive and disabling seizures, despite treatment with maximal doses of AEDs. Adequate treatment with two or more conventional AEDs such as carbamazepine (CBZ), phenytoin (PHT), and phenobarbital (PB) was attempted before presurgical evaluation (36).

Table 1. Summary of clinical findings of patients with ganglioglioma
Total or mean(Range or percentage)
  1. HS, hippocampal sclerosis; CPS, complex partial seizure; SGS, secondarily generalized seizure; SE, status epilepticus; F, frontal; P, parietal; T, temporal; GTR, gross-total resection; PR, partial resection; MSR, mesial structures resection.

Male/female17/13
Mean age at surgery (yr)22.8 (2–55)
LocationFrontal, 5; parietal, 1; temporal, 24
Seizure typeCPS (100%); SGS (23%); SE (3%)
Mean age at seizure onset (yr)10.4 (0.3–22)
Duration of epilepsy (yr)12.5 (1–36)
Extent of resectionGTR (53%); GTR/MSR (33%);
  PR (13%)
HS: present/absent5/25
Balloon cells: present/absent9/21
Postoperative epilepsy:IA (77%); IB (3%);
 Engel's class IIA (17%) IID (3%)

All lesions were surgically treated: complete removal was accomplished in 26 (87%) GGs. The extent of resection was determined by reviewing the operative report and postoperative MRI investigations. In 12 of 24 patients with a temporal lobe lesion, resection included the mesial structures including the amygdala, anterior hippocampus, and the parahippocampal gyrus. We classified the postoperative seizure outcome according to Engel (37). We based our evaluation on a review of the patient files and/or by telephone interview with the patient and family. Follow-up period ranged from 2 to 6 years.

Tissue preparation

Tissue had been fixed in 10% buffered formalin (autopsy tissue, for 2 weeks; surgical specimens, for 12–24 h). Formalin-fixed, paraffin-embedded tissue was sectioned at 6 μm and mounted on organosilane-coated slides (Sigma, St. Louis, MO, U.S.A.). Representative sections of all specimens were processed for hematoxylin–eosin and Nissl stains, as well as for immunocytochemical reactions by using a number of neuronal and glial markers described later. Frozen tissue from control (n = 5) and tumor specimens (n = 5), stored at −80°C, was used for immunocytochemical and Western blot analysis.

Antibody characterization

Glial fibrillary acidic protein (GFAP; polyclonal rabbit, Dako, Glostrup, Denmark; 1:2,000), vimentin (mouse clone V9, Dako; 1:400), synaptophysin (polyclonal rabbit, Dako; 1:200), neuronal nuclear protein (NeuN; mouse clone MAB377, immunoglobulin G (IgG1); Chemicon, Temecula, CA, U.S.A.; 1:1,000) microtubule-associated protein (MAP2; mouse clone HM2, IgG1; Sigma, 1:100; and polyclonal rabbit, Chemicon; 1:1,000) and CD31 (EN-4, IgG1; Sanbio, Uden, The Netherlands; 1:500) were used in the routine immunocytochemical analysis of GGs to document the presence of glial, neuronal, and vascular components of this tumor type. Resting astrocytes were differentiated from reactive astrocytes cells on the basis of morphology and the absence of vimentin immunoreactivity. As previously reported (38,39), vimentin allowed the detection of reactive hypertrophic astrocytes.

For the detection of MVP, two well-characterized monoclonal antibodies (mAbs) MVP-37 (1:50; IgG2b) and LRP-56 (1:25; IgG2b) were used. The characteristics of these mAbs have been described in detail previously (29,40,41).

Immunoblot analysis

For immunoblot analysis, human normal brain (cortex) and tumor samples were homogenized in lysis buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 1% NP-40, 5 mM ethylenediamine tetraacetic acid, and protease inhibitor cocktail (Boehringer Mannheim, Germany). Cell fractionation and sucrose gradient centrifugation were performed as previously described by Berger et al. (42). Protein content was determined by using the bicinchoninic acid method (43). For electrophoresis, equal amounts of proteins (30 μg/lane) were subjected to sodium dodecylsulfate–polyacrylamide gel electrophoretic (SDS-PAGE) analysis. Separated proteins were transferred to nitrocellulose paper for 1 h, by using a semidry electroblotting system (Transblot SD; Bio-Rad, Hercules, CA, U.S.A.), and incubated in TTBS (50 mM Tris-HCl, 0.1% Tween-20, 154 mM NaCl, pH 7.5), containing 5% nonfat dry milk and 1% bovine serum albumin (BSA) for 1 h. Samples were then incubated overnight in TTBS/3% BSA/0.1% sodium azide, containing the primary antibody (MVP-37; 1:500; antiactin, monoclonal mouse, Sigma, 1:1,000). After several washes in TTBS, the membranes were incubated in TTBS/5% nonfat dry milk 1% BSA, containing goat-antimouse Ab coupled to horseradish peroxidase (1:1,500; Dako) for 2 h. After several washes in TTBS, immunoreactive bands were visualized by using an enhanced chemiluminescence kit (Amersham, Buckinghamshire, U.K.). The levels of MVP were evaluated by measuring optical densities of the protein bands by using Scion Image for Windows (beta 4.02) image-analysis software. Data were compared by analysis of variance (ANOVA) with a Fisher Protected Least Significant Difference post hoc analysis (p < 0.05 was defined as statistically significant). Expression of β-actin (as reference protein) was also analyzed in the same protein extracts.

Immunocytochemistry

Sections of routinely processed formalin-fixed, paraffin-embedded tissues were deparaffinated in xylene and, after rinses in ethanol (100% and 95%), were incubated with 1% H2O2 diluted in methanol for 20 min. Slides were then washed with phosphate-buffered saline (PBS; 10 mM, pH 7.4). The slides were placed into sodium citrate buffer (0.01 M, pH 6.0) and heated in a microwave oven (650 W for 10 min; except for GFAP). The slides were allowed to cool for 20 min in the same solution at room temperature and then washed in PBS. They were incubated with a mixture of 10% normal goat serum (NGS) for 1 h before the incubation with the primary Ab (30 min at room temperature and at 4°C overnight). Sections were then washed thoroughly with PBS and incubated at room temperature for 1 h with the appropriate biotinylated secondary Ab diluted in PBS [1:400 goat-antirabbit immunoglobulin (Ig) or 1:200 goat-antimouse Ig (Dako)]. Single-label immunocytochemistry was carried out by using the avidin–biotin peroxidase method (Vector Laboratories, Burlingame, CA, U.S.A.) and 3,3′-diaminobenzidine as chromogen. Sections were counterstained with hematoxylin, dehydrated in alcohol and xylene, and coverslipped. Sections incubated without the primary Ab or with preimmune sera were essentially blank. As positive controls for immunocytochemical staining, paraffin-embedded autopsy specimens of normal human lung tissue were used. Cryosections (5 μm thick) were air-dried overnight and fixed for 10 min in acetone at room temperature before immunostaining with LRP-56. For MVP-37 staining, cryosections were fixed with paraformaldehyde and pretreated with guanidine hydrochloride, as previously described (29).

For double labeling, sections (after incubation with primary Abs; GFAP, NeuN or CD31 and MVP-37) were incubated for 2 days at 4°C with fluorochrome (FITC or Cy3)-conjugated antimouse or antirabbit Ig subclass-specific antibodies (1:200; purchased from either Southern Biotechnology Associates, Inc., Birmingham, AL, or Jackson ImmunoResearch, West Grove, PA, U.S.A.). To block autofluorescence due to the presence of lipofuscin pigment in the tissue, sections were stained with Sudan Black B (Merck) for 10 min, as previously described (44). Sections were then analyzed by means of a laser scanning confocal microscope (Bio-Rad; MRC1024) equipped with an argon-ion laser.

Evaluation of immunostaining

Labeled tissue sections from both types of lesions were examined independently by two observers with respect to the presence or absence of various histopathologic parameters and specific immunoreactivity (IR) for the different markers in neurons, vessels, and glial (or glia–neuronal balloon cells). Two representative paraffin sections per case were stained and assessed with the MVP-37 MVP mAb. The intensity of MVP-immunoreactive staining was stratified into three categories: [0: −, no; 1: +, moderate; 2: ++, strong staining (intensity score)]. The proportion of positive cells or vessels also was stratified into three groups: 1, <10%; 2, 11–40%; 3, >40% (frequency score). Neuronal cell bodies were differentiated from glia and glia–neuronal balloon cells on the basis of morphology, and cells were counted from 10 representative fields of two labeled sections of each tumor at a magnification of × 250, by using an ocular grid as previously described (6,45). Only neurons in which the nucleus could be clearly identified were included. Balloon cells have eccentric nuclei and ballooned opalescent eosinophilic cytoplasm. Sections stained with NeuN, MAP-2, and GFAP adjacent to those used for the MVP staining also were studied. The product of these two values (intensity and frequency scores) was taken to give the overall score (total score).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Case material and histologic features

The 30 surgical specimens from patients with GG were examined. The histopathologic features of these cases are summarized in Table 1. All patients had a history of chronic pharmacoresistant epilepsy. Postoperatively, 23 patients with GG (77%) were completely seizure free (Engel IA). The long-term follow-up of seizure outcome after surgery in glioneuronal tumors (including that of several lesions of the present series) has been presented elsewhere (2). Histologically GGs were composed of a mixture of atypical neuronal cells and neoplastic astrocytes. Cells with abnormal shape and lack of uniform orientation represented the neuronal component. Atypical large and pleomorphic cells with eccentric nuclei and ballooned opalescent eosinophilic cytoplasm (referred to as balloon cells) were observed in nine GGs of our series (Table 1).

Major vault protein expression levels in gangliogliomas

Specific anti-MVP mAb (MVP-37) was used to study the MVP protein expression in gangliogliomas. In protein extracts (total lysate) derived from surgical specimens of GGs, MVP was consistently detectable (as a single band at ∼110 kDa) in all five specimens analyzed, whereas expression in control cortex was low or under the detection level (Fig. 1A). Detection of MVP in cell fractions of GGs (Fig. 1B) indicated predominant localization of MVP in the cytoplasm. Most of the protein was detected in the postnuclear supernatant, pelleted at 100,000 g (P fraction). No detectable expression was observed in the supernatant after the 100,000 g centrifugation step (soluble fraction, S). A small portion was, however, observed in the nucleic fraction (N; Fig. 1B). Total tumor extracts analyzed by sucrose gradient centrifugation (Fig. 1C) demonstrated that MVP is detectable in the 45–50% fractions, which have been previously shown to accumulate intact purified vaults (28). Densitometric analysis of Western blots showed that, by comparison with control cortex, GGs exhibited a significant increase in MVP protein expression (p < 0.05).

image

Figure 1. Expression of major vault protein (MVP) in gangliogliomas. A: Representative immunoblots of MVP in total homogenates from control cortex (CTX) and gangliogliomas (GGs). Proteins (30 μg/lane) were subjected to Western blot analysis with the MVP-37, as described in Materials and Methods. Expression (as reference protein) of β-actin (Actin; 42 kDa) is shown in the same tumor protein extracts. B: Subcellular localization of MVP in GG. Specimens were fractionated into nuclei (N) and 100,000 g particle fraction (P); the remaining supernatant following the 100,000 g centrifugation step was designated soluble fraction (S). C: Fractionation of total GG extracts was carried out by sucrose equilibrium gradient centrifugation. Fractions at the indicated sucrose concentrations were analyzed with Western blot.

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Major vault protein immunoreactivity in normal human brain

Immunocytochemistry using mAbs (MVP-37 and LRP-56) directed against MVP showed no neuronal labeling in control normal human cortex (Fig. 2A; MVP-37). Glial cells in white and gray matter also were negative for MVP, and weak staining was only occasionally observed in blood vessels by using the two MVP mAbs (MVP-37, Fig. 2A and B; Table 2; LRP-56, not shown).

image

Figure 2. Distribution of major vault protein (MVP) protein immunoreactivity in control and ganglioglioma (GG) specimens. Representative photomicrographs of immunohistochemical staining for MVP. Sections are counterstained with hematoxylin (A–J). A, B: MVP in normal brain. No neuronal or glial labeling is observed in control normal cortex (CTX; A) and white matter (Wm; B); only occasionally weak immunoreactivity is detected in blood vessels (arrows). Immunoreactivity for MVP in GGs (C–H). C: Expression in the neuronal (arrows), glial (arrowheads), and vascular components of the tumor (double arrowheads). D: MVP-positive blood vessel (arrows). Neuronal cells of different size are immunoreactive for MVP (arrows in E–G), whereas some neuronal cells (arrowhead in G) appear not labeled. H: MVP-positive balloon cells (arrows) surrounded by positive glial cells (arrowheads); MVP-positive glial cells with di- or multinuclei also were observed (asterisk). I–J: Perilesional cortex adjacent to GG; the perilesional region shows undetectable glial and neuronal MVP staining in both cortex (I) and white matter (J). Weak MVP IR (similar to the staining observed in normal control cortex) is present in blood vessels (arrows). Scale bar: A, I, J, 100 μm; B-H, bar in C, 50 μm.

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Table 2. Major vault protein immunocytochemical expression in different cellular types in control cortex and in cases of ganglioglioma (percentage of cases with immunoreactive cells)
Normal cortex (n = 6)Gangliogliomas
  1. FCD, focal cortical dysplasia; immunoreactivity: −, not present; + moderate; ++ strong.

NeuronsGlial cellsBlood vesselsNeuronsGlial cellsBalloon cellsBlood vessels
   (n = 30)(n = 30)(n = 9)(n = 30)
   − +  ++ −  + ++−  +  ++−  +  ++
−(100%)−(100%)+ (33%)0  60%  40%10%  83%  7%0  56%  44%0  30%  70%

Major vault protein immunoreactivity in gangliogliomas

In contrast to control tissue, clear MVP immunoreactivity was observed in all GG specimens examined (Table 2). All GGs (n = 30) contained neuronal cells with moderate to strong IR for MVP (Table 2; Fig. 2C, E, F, G; Fig. 3A–D). The neuronal labeling index in GGs was >40% in 50% of the cases. In the majority of GGs, moderate MVP IR also was detectable in tumor astrocytes (Fig. 2C, H; Table 2). MVP staining highlighted, however, a variable degree of IR in astroglial cells. Glial labeling indices in 15 of 30 of the cases ranged from 11 to 40%; in 12 cases, the labeling index was <10%, and no glial labeling was observed in three cases. In specimens with low or no detectable glial staining, double labeling with GFAP and NeuN showed a predominant expression in the neuronal component of the tumor (Fig. 3A–D). In all GG cases, tumor vessels displayed MVP labeling (Figs. 2C, D; 3E), and strong vascular IR was observed in 21 of 30 GGs (Table 2). MVP expression also was observed in large ballooned cells in GG specimens containing this cell type (Table 2; Figs. 2H and 3C). MVP labeling was mainly localized in the cytoplasm in both neuronal and glial cells (nuclei remained unstained; Figs. 2 and 3). In agreement with previous studies (30,46,47), a dotted cytoplasmic pattern was often observed. Figure 4 shows the distribution of MVP neuronal, glial, and vascular IR (total) score in control, GG, and peritumoral specimens, indicating that the increased expression was localized within the lesion. No significant differences in MVP protein expression (in terms of intensity of staining and IR cell numbers) were observed between patients with relatively short (<10 years) or long duration of epilepsy (>10 years) or with different clinical outcome.

image

Figure 3. Confocal images of major vault protein (MVP) expression in gangliogliomas (GGs). A–C: Double-labeling of MVP (red) with glial fibrillary acidic protein (GFAP) (green) showing MVP expression in neuronal cells (GFAP-negative) of different size (arrowheads in A and B) and in GFAP-negative ballooned cells. A few astrocytes show colocalization (yellow) of GFAP with MVP (arrows in C). D: Merged image, showing colocalization (yellow) of MVP (red) with NeuN (green) in a group of neurons. E: Merged image, showing localization of MVP (red) in a blood vessel (CD31-positive; green). Scale bar: A–C, 50 μm; D, E, 25 μm.

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image

Figure 4. Major vault protein (MVP) immunoreactivity (IR) scores. A: Plot showing the distribution of MVP IR in controls, gangliogliomas (GGs), and peritumoral specimens. The IR score represents the total score, which was taken as the product of the intensity score and the frequency score. CTX, cortex; N-CTX, normal control CTX; nd, not detectable.

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Major vault protein expression in perilesional regions and hippocampus

As compared with the staining in normal brain, perilesional tissue did not show dramatic changes in the MVP IR pattern. No detectable increase of MVP labeling was observed in blood vessels, neurons, or glial cells in normal cortex adjacent to GGs, despite the long duration of epilepsy (>10 years in cases in which perilesional cortex was available; Figs. 2I and J; 4).

Histologically nonsclerotic hippocampus (non-HS) from GG patients also was analyzed and did not show changes in MVP expression compared with autopsy specimens of normal hippocampus from cases with no history of epilepsy (Fig. 5A). MVP IR was, however, detectable in sclerotic hippocampus (HS; Fig. 5B). MVP immunostaining was detectable in spared big neurons in the dentate hilus and in blood vessels (Fig. 5B). However, only a few positive astrocytes were observed in cases in which diffuse reactive gliosis was present (Fig. 5C).

image

Figure 5. Major vault protein (MVP) immunoreactivity in nonsclerotic and sclerotic hippocampus of ganglioglioma (GG) patients. Sections are counterstained with hematoxylin. A: Nonsclerotic hippocampus (non-HS) without detectable expression of MVP. B, C: Sclerotic hippocampus (HS). MVP immunostaining (B) is observed in hypertrophic hilar neurons (arrows) and in blood vessels (double arrowheads). Only a few positive astrocytic processes were observed (arrow, insert in B), despite the presence of diffuse reactive gliosis, detected with vimentin in an adjacent section (C). Scale bar: 200 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Recent evidence suggests that the general mechanisms of resistance to drug treatment that are active in human cancer patients also may be involved in refractoriness to AEDs (48). To date, the expression pattern and the contribution of transmembrane transporter proteins (P-gp and MRP) to MDR has been widely investigated in several causes of refractory epilepsy (including GGs) (16,17). Resistance to drugs is generally considered to be multifactorial; thus additional molecules such as MVP also are believed to contribute to MDR (22). Whether they also play a role in drug resistance in patients with refractory epilepsy remains to be established. The detection of and the expression pattern of MVP in control and epileptic human brain tissue represent the first step to increase our knowledge about the functions of these proteins, including their potential role in the intrinsic MDR phenotype.

In the present study, we demonstrated that GGs, the most common tumor entity in young patients with medically intractable epilepsy, express high amounts of MVP. Gradient centrifugation analysis indicates that all MVP in GGs assembles in particles behaving as intact vaults. In agreement with previous studies in astrocytic brain tumor cells (30), MVP particles in GGs are located predominantly at cytoplasmic sites. The overexpression and the subcellular location of MVP detected via immunoblotting are confirmed by immunocytochemistry. Expression of MVP was observed in the cytoplasm of neuronal and glial cells of GG specimens. In normal brain, both neurons and resting glial cells do not have detectable expression of MVP. This observation is in agreement with previous studies showing low vault IR in adult brain tissue in both humans and rats (49,50). Whereas adult glial and neuronal cells lack any detectable expression of MVP, strong MVP expression is observed during embryonic and early postnatal development in rat brain (50). Thus reappearance of this embryonic protein in GGs might support the malformative and plastic nature of these tumors. Although the histogenesis of glioneuronal tumors still remains speculative, one possible hypothesis is that they originate from still immature or multipotent dysplastic cells. Recent studies demonstrate the detection in GGs of other proteins, which are expressed early during development and reflect an immature phenotype (3,51,52).

Activation of MVP expression also might occur during the process of malignant transformation of astrocytes. Malignant astrocytes in vitro (cell lines and primary cultures) express high amounts of MVP protein (30). Moreover, a strong expression of MVP has been reported in both tumor glial cells and neoplastic vessels of surgical specimens from patients with glioblastomas (53). Accordingly, we also observed increased expression of MVP in the vascular compartment of GGs. Expression of MVP in blood vessels has been observed during development in rat brain (50). Whether MVP is expressed exclusively by vascular endothelial cells (47) or also by perivascular pericytes (50) is still unclear. Because no single commonly used marker identifies all pericytes with certainty, it is difficult to identify pericytes in pathologic conditions, such as cancer, when these cells change their expression of marker proteins (54). Immunogold electron-microscopic studies (in non–paraffin-embedded material) will be necessary to define the cellular localization of MVP within blood vessels. Recently increased expression of other MRPs (multidrug transporter proteins, MRP1 and P-gp) was observed in the vascular compartment of glioneuronal lesions (17). This is of particular interest in relation to their function as outwardly directed efflux pumps, which may limit the brain accumulation of different lipophilic drugs, including AEDs. The different physiologic functions and the functional consequences of the increased expression of MRPs at the level of the blood–brain and blood–tumor barriers in drug-resistant human epilepsy remain to be determined.

Interestingly, the overexpression of MVP labeling was observed in the lesion and not in perilesional areas. This observation supports the hypothesis of constitutive rather than induced or acquired expression and is in keeping with the previously suggested role for the transmembrane transporter protein overexpression observed in different developmental lesions associated with refractory epilepsy (16,17). It is not likely that treatment with AEDs per se is responsible for the observed intralesional MVP overexpression, because tissue adjacent to the lesion has been theoretically exposed to the same drugs. The potential interactions of vaults with AEDs, as well as the influence of AED exposure on MVP expression, should be investigated further in normal and tumor astrocytes expressing MVP protein in vitro.

We cannot totally exclude that the overexpression in epileptogenic brain tissue is related to seizure activity. MVP expression was detectable in sclerotic hippocampus from patients with dual pathology. Association of GGs with mesial temporal sclerosis (MTS) has been previously described and may define a distinct pathophysiologic category of MTS (55,56). Strong MVP expression was observed in hypertrophic cells in the dentate hilus. Hypethrophic cells within the dentate hilus and similar overexpression of MVP also were observed in adult patients with temporal lobe epilepsy [TLE; (57) Sisodiya et al., unpublished observation]. Activation of MVP expression may represent a feature of hippocampal pathology in TLE. In our study, with fixed material, it was not possible to investigate the spatiotemporal development or the functional consequences of this overexpression. For this purpose, the use of experimental models of pharmacoresistant epilepsy is required.

Although the cellular function of vault has not been completely clarified, tissue-distribution studies point to a protective role against toxic compounds as for other MRPs [P-gp and MRP; (22)]. Overexpression of MVP [together with P-gp and MRP1; (17)] may function as a general protection mechanism, which could influence the fate and the survival of cells expressing this phenotype early during development. Moreover, overexpression of MVP in the epileptogenic neuronal component of GGs also may affect the response to AEDs, by changing the subcellular compartmentalization of drugs (22).

These results [together with previous observations in GGs (17)], indicate that drug resistance–related proteins (P-gp, MRP1, and MVP) are regulated in concert in GGs. MDR in these lesions may be the result of different mechanisms operating at different levels. Expression of drug resistance–associated proteins at the level of the blood–brain and blood–tumor barriers may impair the penetration of therapeutic agents, leading to decreased tissue drug concentrations. Moreover, activation of expression at the level of the neuronal and neuroglial epileptogenic component of the GGs may interfere with the intracellular activities of the drugs by compartmentalization of drugs away from the intracellular target (MVP) or pump molecules (P-gp, MRP1). Thus evaluation of the role of vault molecules in the sequestration and compartmentalization of AEDs might be worthwhile and requires careful consideration.

Acknowledgments

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Acknowledgment:  This work was supported by the “Christelijke Vereniging voor de Verpleging van Lijders aan Epilepsie” (E.A. van Vliet and E. Aronica), the Stichting AZUA-funds (E. Aronica), the National Epilepsy Fund: “Power of the Small,” and Hersenstichting Nederland (J.A. Gorter; E. Aronica, NEF grant 02-10).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES
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