- Email: [email protected]
Multidrug resistance proteins in tuberous sclerosis and refractory epilepsy
Multidrug Resistance Proteins in Tuberous Sclerosis and Refractory Epilepsy Alberto Lazarowski, PhD*†, Fabiana Lubieniecki, MD‡, Sandra Camarero, HT‡, Hugo Pomata, MD§, Marcelo Bartuluchi, MD§, Gustavo Sevlever, MD, PhD㥋, and Ana Lı´a Taratuto, MD, PhD‡㥋 Tuberous sclerosis is an autosomal dominant syndrome characterized by seizures that are refractory to medication in severely affected individuals. The mechanism involved in drug resistance in tuberous sclerosis is unknown. The proteins MDR-1 (multidrug resistance) and MRP-1 (multidrug resistance-associated protein-1) are linked to chemotherapy resistance in tumor cells. However, the relationship between refractoriness to antiepileptic drugs and MDR-1 or MRP-1 brain expression has been poorly studied. We have previously described a case of tuberous sclerosis with refractory epilepsy that expressed multidrug resistance gene (MDR-1) in tuber cells from epileptogenic brain lesion. In this retrospective study, we describe the expression of MDR-1 and MRP-1 in the epileptogenic cortical tubers of three pediatric patients with tuberous sclerosis and refractory epilepsy surgically treated. Monoclonal antibodies for MDR-1 and MRP-1 proteins were used for immunohistochemistry. In epileptogenic cortical tuber brain specimens, MDR-1 and MRP-1 proteins were strongly immunoreactive in abnormal balloon cells, dysplastic neurons, astrocytes, microglial cells, and some blood-brain vessels. A more extensive MDR-1 immunoreactivity was observed. These data suggest that refractory epilepsy phenotype in tuberous sclerosis can be associated with the expression of both multidrug resistance MDR-1 and MRP-1 transporters in epileptogenic cortical tubers. © 2004 by Elsevier Inc. All rights reserved. Lazarowski A, Lubieniecki F, Camarero S, Pomata H, Bartuluchi M, Sevlever G, Taratuto AL. Multidrug resistance proteins in tuberous sclerosis and refractory epilepsy. Pediatr Neurol 2004;30:102-106.
From the *Clinical Biochemistry Department, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires (UBA); †Institute of Cell Biology and Neurobiology “Dr. E. de Robertis”, Facultad de Medicina, UBA; ‡Laboratory of Neuropathology, Hospital Nacional de Pediatria “Prof. Dr. Juan P. Garrahan”; §Neurosurgery Department, Hospital Nacional de Pediatria “Prof. Dr. Juan P. Garrahan”; and 㛳 Laboratory of Neuropathology, Instituto de Investigaciones Neurologicas “Dr. Raul Carrea” FLENI, Buenos Aires, Argentina.
102
PEDIATRIC NEUROLOGY
Vol. 30 No. 2
Introduction Tuberous sclerosis is a multisystemic disorder that primarily affects the brain, skin, and kidney. Hamartin and tuberin are the products of the TSC1 and TSC2 tumor suppressor genes, respectively. Within the last few years, mutations in these genes have been identified as responsible for this disease [1,2], and there is evidence to suggest that the TSC2 product, tuberin, suppresses tumorigenicity [3,4]. Mutations of p53 tumor suppressor gene have been related with refractoriness to chemotherapy in patients with cancer in whom the expression of MDR-1 and MRP-1 genes was also demonstrated [5]. MDR-1 and MRP-1 transporters are highly expressed in tumor cells of patients undergoing anticancer chemotherapy, which results in multidrug resistance to cytotoxic agents [6-8]. The mechanism involved in drug resistance in cases of tuberous sclerosis remains to be elucidated. Cortical tubers are important features of the disease. Selected drugresistant patients with epileptogenic cortical tubers could be considered for epilepsy surgery [9-12]. In a previous report, we described a potential association of tuberous sclerosis and refractory epilepsy with the expression of MDR-1 gene in tuber cells in an epileptogenic brain lesion [13]. The high expression of MDR-1 gene in tuber cells observed in that first case report could be an important hallmark of this pathology. Because it was a single isolated report, the investigation of further similar tuberous sclerosis cases with refractory epilepsy will yield useful information in this regard. In the present retrospective study, we describe the expression of multidrug resistance MDR-1 and MRP-1 proteins in balloon cells, dysplastic neurons, astrocytes,
Communications should be addressed to: Dr. Lazarowski; Caseros 1944 #9B; (1152) Buenos Aires, Argentina. Received February 25, 2003; accepted May 30, 2003.
© 2004 by Elsevier Inc. All rights reserved. doi:10.1016/S0887-8994(03)00407-7 ● 0887-8994/04/$—see front matter
Table 1.
MDR-1 and MRP-1 immunostaining pattern in brain of three cases of tuberous sclerosis
Cases 1
Astrocytes 2
3
Large Neurons 1 2 3
1
Balloon Cells 2
3
1
Vessels 2
Normal Tissue 3
MDR-1*
⫹
⫹
⫹⫹
⫹
⫹
⫹
⫹
⫹⫹
⫹
⫹
⫹
⫺/⫹
MRP-1** Synaptophysin NF*
⫺ ⫺ ⫺
⫺/⫹ ⫺ ⫺
⫹ ⫺ ⫺
⫹ ⫹ ⫹
⫹ ⫹ ⫹
⫹ ⫹ ⫹
⫹ ⫺/⫹ ⫺/⫹
⫹ ⫺/⫹ ⫹
⫹/⫺ ⫺/⫹ ⫺
⫺
⫺
⫺/⫹
GFAP*
⫹
⫹
⫹
⫺
⫺
⫺
⫺/⫹
⫺
⫺/⫹
Few vessels and glia cells ⫹/⫺ ⫺ ⫹ Isolated “normal neurons” ⫹
* The intensity of the reaction was variable in different areas within the abnormal tissue and in the surrounding cerebral cortex. ** Scattered slight positive cells. Abbreviations: GFAP ⫽ Glial fibrillary acidic protein MDR-1 ⫽ Multidrug resistance 1 MRP-1 ⫽ Multidrug resistance–associated protein 1 NF ⫽ Neurofilament
and microglial cells from three pediatric patients with tuberous sclerosis surgically treated for their refractory epilepsy.
white matter. Silver impregnation revealed dysmorphic and giant neuronal perikaryon and dendritic and axonal processes, whereas balloon cells appeared round with a yellow cytoplasm. Reactive astrocytes were also observed.
Materials and Methods Immunohistochemistry Our patients manifesting complete diagnostic criteria of tuberous sclerosis with uncontrollable seizures [9-12] were surgically treated, and their brain specimens were retrospectively studied, as described: Brain tissue samples: Surgical specimens of brain tissues were selected from the tissue collection of the Pathology Laboratory of the Garrahan Children’s Hospital of Buenos Aires, on the basis of the pathologic diagnosis of cortical tubers from three pediatric patients with tuberous sclerosis and refractory epilepsy. Morphologic analysis: Brain tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin, Luxol-Fast-Blue with periodic acid-Schiff (PAS), and Bielschowsky method for morphologic analysis. Immunohistochemistry: The monoclonal antibodies and dilutions were used as follows: MDR-1 antibody (1:50, Clone JSB-1, Novocastra, UK), MRP-1 antibody (1:50, Clone QCRL1, Signet, Dedham, MA), and Neurofilament (1:70, Clone 2F11), glial fibrillary acidic protein (1:50, Clone 6F2), and Synaptophysin (1:20, Clone Sy38) from Dako (Capinteria, CA). Secondary polyclonal antibody for the immunoperoxidase technique (Sigma) and the LSAB2 detection system (Dako) were used. Thin sections of kidney and liver were used as immunostaining positive control specimens for MDR-1 and MRP-1 expression; the specimens were collected from the same tissue archives (not shown).
Results Morphologic Description Tissue sections representative of cerebral cortex and associated white matter were used in these studies. Abnormal cells, including large neurons and balloon cells, as well as neuronal and glial cells with normal morphology were present in most sections. Abnormal cortical lamination, neuronal dysmorphisms with giant neurons, as well as a variable number of large and eosinophilic cells (balloon cells) similar to focal cortical dysplasia balloon cells were present in all cortical layers and subcortical
Immunohistochemical analysis yielded the following results: Glial fibrillary acidic protein: See Table 1. Neurofilament and Synaptophysin: Neuronal markers immunostaining ranged from positive balloon cells with granular cytoplasmatic staining, to extremely attenuated or negative. MDR-1 and MRP-1: Immunostaining for MDR-1 and MRP-1 was observed in varying amounts in dysplastic neurons, balloon cells (Fig 1A and Fig 2A), astrocytes, and microglial cells (Fig 1B and Fig 2B). MDR-1 was also immunoreactive in some vessels, whereas others remained unstained. Slightly positive immunostaining for MRP-1 in vessels was disclosed in only one case (Table 1). Discussion Tuberous sclerosis is a disorder of cell migration, proliferation, and differentiation that involves various neurologic phenotypes including epilepsy in 80-96% of patients. Seizures often begin in the first month of life and are frequently severe and intractable [10-12]. We previously described the MDR-1 gene product expression in tuber cells in an epileptogenic brain lesion in a patient with tuberous sclerosis surgically treated for refractory epilepsy [13]. Recently the presence of MDR-1 or MRP-1 proteins was demonstrated in brain parenchymal cells from patients with the three common causes of refractory epilepsy— dysembryoplastic neuroepithelial tumors, focal cortical dysplasias, and hypocampal sclerosis—and in these cases a constitutive expression of the multidrug transporters has
Lazarowski et al: MDR-1 and MRP-1 in Tuberous Sclerosis 103
Figure 1. MDR-1 immunostaining. (A) Positive balloon cells (arrow) (400⫻). (B) Positive microglial cell (arrow) (400⫻).
Figure 2. MRP-1 immunostaining. (A) Positive (arrow) and negative (ⴱ) balloon cells (400⫻). (B) Positive microglial cell (arrow) and negative balloon cells (ⴱ) (250⫻).
been proposed [14]. To date, few clinical reports suggest a role for brain expression of the multidrug transporters MDR-1 or MRP-1 in parenchymal nonvascular endothelial cells in relationship to the development of refractory epilepsy [13-15]. Our results indicate the detection of both multidrug resistance proteins in abnormal balloon cells and dysplastic neurons, and also in other parenchymal brain cells (Figs 1 and 2). These results differ from previous descriptions [14] because both multidrug resistance proteins were present in the same brain epileptic lesion from these tuberous sclerosis cases. We also observed that some balloon cells were negative for MDR-1 and some were negative for MRP-1 in separate staining procedures; it is unknown whether the negative balloon cells for one transporter could be positive for the other one. The expression of the MDR-1 gene was more intense and extensive than MRP-1 in all three cases presented here (Table 1). These differences could be partly due to seizure-induced expression for the MDR-1 gene according to results recently described in some experimental epilepsy models [16-21]. The seizure-inducible mechanism for MRP-1 gene upregulation still remains to be demonstrated. The hamartin-tuberin complex activity as tumor suppressor gene has been documented, but its relationship
with multidrug transporter expression has not yet been investigated. However, a role for another tumor suppressor gene (p53) in multidrug resistance phenotype induction has been described in cancer cells. Extensive studies suggest that MDR-1 and MRP-1 gene expression is closely related to transcriptional suppression by p53 protein. Both MDR-1 and MRP-1 products are expressed in tumors that frequently fail to express wildtype p53 [22-27]. Some regulatory mechanisms have been suggested [28,29]. To date, few isolated reports have demonstrated absent or mutant p53 expression in tuberous sclerosis–related tumor cells [30-33]. It has been recently reported that tuberous sclerosiscomplex activity is linked with rapamycin receptor (mTOR) downstream signaling [34-36]. The same mTORdependent activity has also been linked with drug resistance in prostate cancer cells [37] and control of the upregulation of hypoxia inducible factor (HIF-1␣) [38], closely related to tumor progression [39]. HIF-1␣ is also a promoter of MDR-1 gene expression [40]. These observations suggest that one HIF-1␣-dependent pathway could be active and inducing the upregulation of MDR-1 gene expression in tuberous sclerosis complex mutant cells through nonrepressed mTOR function.
104
PEDIATRIC NEUROLOGY
Vol. 30 No. 2
Figure 3. Schematic representation of MDR-1 and MRP-1 transporters.
In tuberous sclerosis, either of the alternative mechanisms described above could occur according to the hypothesis of a “constitutive expression” of multidrug transporters in cortical tuber abnormal cells, as proposed by Sisodiya et al. [14] in brain developmental malformations with refractory epilepsy. Our current and previous results are the only available investigations related to multidrug resistance protein expression in cortical tuber cells from epileptogenic brain lesions of patients with tuberous sclerosis; however, whether the TSC1 or TSC2 genes play any regulatory role in MDR-1 or MRP-1 gene expression still remains to be elucidated. Hypothetically, we could assume that the abnormal nature of balloon cells and dysplastic neurons evident in cortical tubers are important features that could support the presence of MDR-1 or MRP-1 in such cells as a constitutive expression of these genes. However, the presence of MDR-1 and MRP-1 proteins observed in some normal cells from the same cortical tuber lesions of our patients is in agreement with a potential mechanism of inducible expression of the multidrug resistance proteins, likely secondary to persistent and repetitive uncontrolled seizures, as demonstrated in experimental epileptic models. We believe that both mechanisms may play a critical role in the earlier development of the refractory epilepsy phenotype in patients with tuberous sclerosis. Functionally, the MDR-1 protein has been shown to elicit extrusion of a variety of hydrophobic, often cationic or neutral substrates, immunosuppressant drugs including rapamycin [41], and also planar lipophilic molecular structures, which are the most common structures of the antiepileptic drugs currently used [42]. MRP-1 protein acts as a transporter of several anionic residues of exogenous or endogenous compounds, and conjugates with glutathione, or epoxides and glucuronides, which are the major metabolic pathway of carbamazepine and lamotrigine [42]. The pattern observed of highly positive immunostaining for MDR-1 and MRP-1 proteins, in different cells of the epileptogenic cortical tubers, may result in a wide spec-
trum of refractory cells as a “pharmacoresistant cellular network” playing a critical role in the development of refractory epilepsy in tuberous sclerosis. The sum of functional properties of both proteins can confer a higher multidrug resistance phenotype to tuberous sclerosis (Fig 3). These results do not explain why the cortical tuber lesions are epileptogenic [12], but perhaps they may help to elucidate why these epileptogenic cortical tubers are refractory to antiepileptic drugs. Our results suggest that brain expression of both MDR-1 and MRP-1 transporters in the epileptogenic cortical tuber can be related to the refractory epilepsy phenotype observed in tuberous sclerosis. References [1] van Slegtenhorst M, Nellist M, Nagelkerken B, et al. Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products. Hum Mol Genet 1998;7:1053-7. [2] The European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of tuberous sclerosis gene on chromosome 16. Cell 1993;75:1305-15. [3] Jin F, Wienecke R, Xiao GH, Maize JC, Jr, DeClue JE, Yeung RS. Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region. Proc Natl Acad Sci USA 1996;93:9154-9. [4] Orimoto K, Tsuchiya H, Kobayashi T, Matsuda T, Hino O. Suppression of the neoplastic phenotype by replacement of the Tsc2 gene in Eker rat renal carcinoma cells. Biochem Biophys Res Commun 1996;219:70-5. [5] Bush JA, Li G. Cancer chemoresistance: The relationship between p53 and multidrug transporters. Int J Cancer 2002;98:323-30. [6] Juliano RL, Ling VA. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochem Biophys Acta 1976;445:152-62. [7] Gottesman MM, Pastan I. The multidrug transporter, a doubleedge sword. J Biol Chem 1988;263:12163-6. [8] Cole SP, Deeley RG. Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP. Bioessays 1998;20: 931-40 [Review]. [9] McClintock WM. Neurologic manifestations of tuberous sclerosis complex. Curr Neurol Neurosci Rep 2002;158-63 [Review]. [10] Curatolo P, Verdecchia M, Bombardieri R. Tuberous sclerosis complex: A review of neurological aspects. Eur J Paediatr Neurol 2002;6:15-23.
Lazarowski et al: MDR-1 and MRP-1 in Tuberous Sclerosis 105
[11] Avellino AM, Berger MS, Rostomily RC, Shaw CM, Ojemann GA. Surgical management and seizure outcome in patients with tuberous sclerosis. J Neurosurg 1997;87:391-6. [12] Crino PB, Miyata H, Vinters HV. Neurodevelopmental disorders as a cause of seizures: Neuropathologic, genetic, and mechanistic considerations. Brain Pathol 2002;12:212-33. [13] Lazarowski A, Sevlever G, Taratuto A, Massaro M, Rabinowicz A. Tuberous sclerosis associated with MDR1 gene expression and drug-resistant epilepsy. Ped Neurol 1999;21:731-4. [14] Sisodiya SM, Lin WR, Harding BN, Squier MV, Thom M. Drug resistance in epilepsy: Expression of drug resistance proteins in common cause of refractory epilepsy. Brain 2002;125:22-31. [15] Tishler DM, Weinberg KI, Hinton DR, Barbaro N, Annett GM, Raffel C. MDR1 gene expression in brain of patients with medically intractable epilepsy. Epilepsia 1995;36:1-6. [16] Zhang L, Ong W, Lee T. Induction of P-glycoprotein expression in astrocytes following intracerebroventricular kainate injection. Exp Brain Res 1999;126:509-16. [17] Lazarowski A, Girardi E, Ramos A, Garcı´a-Rivello H, Brusco A. MDR-1 gene expression (Pgp-170) in different brain areas in an experimental epilepsy model [Abstract]. J Epilepsy Clin Neurophysiol 2002;8:101. [18] Seegers U, Potschka H, Lo¨scher W. Expression of the multidrug transporter P-glycoprotein in brain capillary endothelial cells and brain parenchyma of amygdala-kindled rats. Epilepsia 2002;43:675-84. [19] Rizzi M, Caccia S, Guisso G, et al. Limbic seizures induce P-glycoprotein in rodent brain: Functional implications for pharmacoresistance. J Neurosci 2002;22:5833-9. [20] Kwan P, Sills GJ, Butler E, Gant TW, Meldrum BS, Brodie MJ. Regional expression of multidrug resistance genes in genetically epilepsy-prone rat brain after a single audiogenic seizure. Epilepsia 2002;43: 1318-23. [21] Lazarowski A, Girardi E, Ramos AJ, Garcı´a-Rivello H, Brusco A. Neuronal MDR-1 gene encoded P-glycoprotein (P-170) expression in 3-mercaptopropionic acid-induced seizures in rats. Epilepsia 2002; 43(Suppl. 7):11-12. [22] Sullivan GF, Yang JM, Vassil A, Yang J, Bash-Babula J, Hait WN. Regulation of expression of the multidrug resistance protein MRP1 by p53 in human prostate cancer cells. J Clin Invest 2000;105:1261-7. [23] Fukushima Y, Oshika Y, Tokunaga T, et al. Multidrug resistance-associated protein (MRP) expression is correlated with expression of aberrant p53 protein in colorectal cancer. Eur J Cancer 1999;35:935-8. [24] Oka M, Kounoura K, Narasaki F, et al. P-glycoprotein is positively correlated with p53 protein accumulation in human colorectal cancers. Jpn J Cancer Res 1997;88:738-42. [25] Thottassery JV, Zambetti GP, Arimori K, Schuetz EG, Schuetz JD. p53-dependent regulation of MDR1 gene expression causes selective resistance to chemotherapeutic agents. Proc Natl Acad Sci USA 1997; 94:11037-42. [26] Linn SC, Honkoop AH, Hoekman K, van der Valk P, Pinedo HM, Giaccone G. p53 and P-glycoprotein are often co-expressed and are
106
PEDIATRIC NEUROLOGY
Vol. 30 No. 2
associated with poor prognosis in breast cancer. Br J Cancer 1996;74: 63-8. [27] Ralhan R, Swain RK, Agarwal S, et al. P-glycoprotein is positively correlated with p53 in human oral pre-malignant and malignant lesions and is associated with poor prognosis. Int J Cancer 1999;84:80-5. [28] Sampath J, Sun D, Kidd VJ, et al. Mutant p53 cooperates with ETS and selectively up-regulates human MDR1 not MRP1. J Biol Chem 2001;276:39359-67. [29] Zhan M, Yu D, Lang A, Li L, Pollock RE. Wild type p53 sensitizes soft tissue sarcoma cells to doxorubicin by down-regulating multidrug resistance-1 expression. Cancer 2001;92:1556-66. [30] Kawaguchi K, Oda Y, Nakanishi K, et al. Malignant transformation of renal angiomyolipoma: A case report. Am J Surg Pathol 2002;26:523-9. [31] Kim SK, Wang KC, Cho BK, et al. Biological behavior and tumorigenesis of subependymal giant cell astrocytomas. J Neurooncol 2001;52:217-25. [32] Lantuejoul S, Ferretti G, Negoescu A, Parent B, Brambilla E. Multifocal alveolar hyperplasia associated with lymphangioleiomyomatosis in tuberous sclerosis. Histopathology 1997;30:570-5. [33] Cocker HA, Pinkerton CR, Kelland LR. Characterization and modulation of drug resistance of human paediatric rhabdomyosarcoma cell line. Br J Cancer 2000;83:338-45. [34] Kenerson HL, Aicher LD, True LD, Yeung RS. Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. Cancer Res 2002;62:5645-50. [35] Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC, Blenis J. Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)mediated downstream signaling. Proc Natl Acad Sci USA 2002;99: 13571-6. [36] Onda H, Crino PB, Zhang H, et al. Tsc2 Null murine neuroepithelial cells are a model for human tuber giant cells, and show activation of an mTOR pathway. Mol Cell Neurosci 2002;21:561-74. [37] Grunwald V, DeGraffenried L, Russel D, Friedrichs WE, Ray RB, Hidalgo M. Inhibitors of mTOR reverse doxorubicin resistance conferred by PTEN status in prostate cancer cells. Cancer Res 2002;62: 6141-5. [38] Hudson C, Liu M, Chiang G, et al. Regulation of hypoxiainducible factor 1␣ expression and function by the mammalian target of rapamycin. Mol Cell Biol 2002;22:7004-14. [39] Semenza G. HIF-1 and tumor progression: Pathophysiology and therapeutics. Trends Mol Med 2002;8(Suppl. 4):S62-7 [Review]. [40] Comerford K, Wallace T, Karhausen J, et al. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res 2002;62:3387-94. [41] Sacyshyn BR, Bowen-Yacyshyn MB, Pilasky LM. Inhibition by rapamycyn of P-glycoprotein 170-mediated export from normal lymphocytes. Scand J Immunol 1996;43:449-55. [42] Levy RH, Mattson RH, Meldrum BS. Antiepileptic drugs, 4th ed. New York: Raven Press, 1995.
From the *Clinical Biochemistry Department, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires (UBA); †Institute of Cell Biology and Neurobiology “Dr. E. de Robertis”, Facultad de Medicina, UBA; ‡Laboratory of Neuropathology, Hospital Nacional de Pediatria “Prof. Dr. Juan P. Garrahan”; §Neurosurgery Department, Hospital Nacional de Pediatria “Prof. Dr. Juan P. Garrahan”; and 㛳 Laboratory of Neuropathology, Instituto de Investigaciones Neurologicas “Dr. Raul Carrea” FLENI, Buenos Aires, Argentina.
102
PEDIATRIC NEUROLOGY
Vol. 30 No. 2
Introduction Tuberous sclerosis is a multisystemic disorder that primarily affects the brain, skin, and kidney. Hamartin and tuberin are the products of the TSC1 and TSC2 tumor suppressor genes, respectively. Within the last few years, mutations in these genes have been identified as responsible for this disease [1,2], and there is evidence to suggest that the TSC2 product, tuberin, suppresses tumorigenicity [3,4]. Mutations of p53 tumor suppressor gene have been related with refractoriness to chemotherapy in patients with cancer in whom the expression of MDR-1 and MRP-1 genes was also demonstrated [5]. MDR-1 and MRP-1 transporters are highly expressed in tumor cells of patients undergoing anticancer chemotherapy, which results in multidrug resistance to cytotoxic agents [6-8]. The mechanism involved in drug resistance in cases of tuberous sclerosis remains to be elucidated. Cortical tubers are important features of the disease. Selected drugresistant patients with epileptogenic cortical tubers could be considered for epilepsy surgery [9-12]. In a previous report, we described a potential association of tuberous sclerosis and refractory epilepsy with the expression of MDR-1 gene in tuber cells in an epileptogenic brain lesion [13]. The high expression of MDR-1 gene in tuber cells observed in that first case report could be an important hallmark of this pathology. Because it was a single isolated report, the investigation of further similar tuberous sclerosis cases with refractory epilepsy will yield useful information in this regard. In the present retrospective study, we describe the expression of multidrug resistance MDR-1 and MRP-1 proteins in balloon cells, dysplastic neurons, astrocytes,
Communications should be addressed to: Dr. Lazarowski; Caseros 1944 #9B; (1152) Buenos Aires, Argentina. Received February 25, 2003; accepted May 30, 2003.
© 2004 by Elsevier Inc. All rights reserved. doi:10.1016/S0887-8994(03)00407-7 ● 0887-8994/04/$—see front matter
Table 1.
MDR-1 and MRP-1 immunostaining pattern in brain of three cases of tuberous sclerosis
Cases 1
Astrocytes 2
3
Large Neurons 1 2 3
1
Balloon Cells 2
3
1
Vessels 2
Normal Tissue 3
MDR-1*
⫹
⫹
⫹⫹
⫹
⫹
⫹
⫹
⫹⫹
⫹
⫹
⫹
⫺/⫹
MRP-1** Synaptophysin NF*
⫺ ⫺ ⫺
⫺/⫹ ⫺ ⫺
⫹ ⫺ ⫺
⫹ ⫹ ⫹
⫹ ⫹ ⫹
⫹ ⫹ ⫹
⫹ ⫺/⫹ ⫺/⫹
⫹ ⫺/⫹ ⫹
⫹/⫺ ⫺/⫹ ⫺
⫺
⫺
⫺/⫹
GFAP*
⫹
⫹
⫹
⫺
⫺
⫺
⫺/⫹
⫺
⫺/⫹
Few vessels and glia cells ⫹/⫺ ⫺ ⫹ Isolated “normal neurons” ⫹
* The intensity of the reaction was variable in different areas within the abnormal tissue and in the surrounding cerebral cortex. ** Scattered slight positive cells. Abbreviations: GFAP ⫽ Glial fibrillary acidic protein MDR-1 ⫽ Multidrug resistance 1 MRP-1 ⫽ Multidrug resistance–associated protein 1 NF ⫽ Neurofilament
and microglial cells from three pediatric patients with tuberous sclerosis surgically treated for their refractory epilepsy.
white matter. Silver impregnation revealed dysmorphic and giant neuronal perikaryon and dendritic and axonal processes, whereas balloon cells appeared round with a yellow cytoplasm. Reactive astrocytes were also observed.
Materials and Methods Immunohistochemistry Our patients manifesting complete diagnostic criteria of tuberous sclerosis with uncontrollable seizures [9-12] were surgically treated, and their brain specimens were retrospectively studied, as described: Brain tissue samples: Surgical specimens of brain tissues were selected from the tissue collection of the Pathology Laboratory of the Garrahan Children’s Hospital of Buenos Aires, on the basis of the pathologic diagnosis of cortical tubers from three pediatric patients with tuberous sclerosis and refractory epilepsy. Morphologic analysis: Brain tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin, Luxol-Fast-Blue with periodic acid-Schiff (PAS), and Bielschowsky method for morphologic analysis. Immunohistochemistry: The monoclonal antibodies and dilutions were used as follows: MDR-1 antibody (1:50, Clone JSB-1, Novocastra, UK), MRP-1 antibody (1:50, Clone QCRL1, Signet, Dedham, MA), and Neurofilament (1:70, Clone 2F11), glial fibrillary acidic protein (1:50, Clone 6F2), and Synaptophysin (1:20, Clone Sy38) from Dako (Capinteria, CA). Secondary polyclonal antibody for the immunoperoxidase technique (Sigma) and the LSAB2 detection system (Dako) were used. Thin sections of kidney and liver were used as immunostaining positive control specimens for MDR-1 and MRP-1 expression; the specimens were collected from the same tissue archives (not shown).
Results Morphologic Description Tissue sections representative of cerebral cortex and associated white matter were used in these studies. Abnormal cells, including large neurons and balloon cells, as well as neuronal and glial cells with normal morphology were present in most sections. Abnormal cortical lamination, neuronal dysmorphisms with giant neurons, as well as a variable number of large and eosinophilic cells (balloon cells) similar to focal cortical dysplasia balloon cells were present in all cortical layers and subcortical
Immunohistochemical analysis yielded the following results: Glial fibrillary acidic protein: See Table 1. Neurofilament and Synaptophysin: Neuronal markers immunostaining ranged from positive balloon cells with granular cytoplasmatic staining, to extremely attenuated or negative. MDR-1 and MRP-1: Immunostaining for MDR-1 and MRP-1 was observed in varying amounts in dysplastic neurons, balloon cells (Fig 1A and Fig 2A), astrocytes, and microglial cells (Fig 1B and Fig 2B). MDR-1 was also immunoreactive in some vessels, whereas others remained unstained. Slightly positive immunostaining for MRP-1 in vessels was disclosed in only one case (Table 1). Discussion Tuberous sclerosis is a disorder of cell migration, proliferation, and differentiation that involves various neurologic phenotypes including epilepsy in 80-96% of patients. Seizures often begin in the first month of life and are frequently severe and intractable [10-12]. We previously described the MDR-1 gene product expression in tuber cells in an epileptogenic brain lesion in a patient with tuberous sclerosis surgically treated for refractory epilepsy [13]. Recently the presence of MDR-1 or MRP-1 proteins was demonstrated in brain parenchymal cells from patients with the three common causes of refractory epilepsy— dysembryoplastic neuroepithelial tumors, focal cortical dysplasias, and hypocampal sclerosis—and in these cases a constitutive expression of the multidrug transporters has
Lazarowski et al: MDR-1 and MRP-1 in Tuberous Sclerosis 103
Figure 1. MDR-1 immunostaining. (A) Positive balloon cells (arrow) (400⫻). (B) Positive microglial cell (arrow) (400⫻).
Figure 2. MRP-1 immunostaining. (A) Positive (arrow) and negative (ⴱ) balloon cells (400⫻). (B) Positive microglial cell (arrow) and negative balloon cells (ⴱ) (250⫻).
been proposed [14]. To date, few clinical reports suggest a role for brain expression of the multidrug transporters MDR-1 or MRP-1 in parenchymal nonvascular endothelial cells in relationship to the development of refractory epilepsy [13-15]. Our results indicate the detection of both multidrug resistance proteins in abnormal balloon cells and dysplastic neurons, and also in other parenchymal brain cells (Figs 1 and 2). These results differ from previous descriptions [14] because both multidrug resistance proteins were present in the same brain epileptic lesion from these tuberous sclerosis cases. We also observed that some balloon cells were negative for MDR-1 and some were negative for MRP-1 in separate staining procedures; it is unknown whether the negative balloon cells for one transporter could be positive for the other one. The expression of the MDR-1 gene was more intense and extensive than MRP-1 in all three cases presented here (Table 1). These differences could be partly due to seizure-induced expression for the MDR-1 gene according to results recently described in some experimental epilepsy models [16-21]. The seizure-inducible mechanism for MRP-1 gene upregulation still remains to be demonstrated. The hamartin-tuberin complex activity as tumor suppressor gene has been documented, but its relationship
with multidrug transporter expression has not yet been investigated. However, a role for another tumor suppressor gene (p53) in multidrug resistance phenotype induction has been described in cancer cells. Extensive studies suggest that MDR-1 and MRP-1 gene expression is closely related to transcriptional suppression by p53 protein. Both MDR-1 and MRP-1 products are expressed in tumors that frequently fail to express wildtype p53 [22-27]. Some regulatory mechanisms have been suggested [28,29]. To date, few isolated reports have demonstrated absent or mutant p53 expression in tuberous sclerosis–related tumor cells [30-33]. It has been recently reported that tuberous sclerosiscomplex activity is linked with rapamycin receptor (mTOR) downstream signaling [34-36]. The same mTORdependent activity has also been linked with drug resistance in prostate cancer cells [37] and control of the upregulation of hypoxia inducible factor (HIF-1␣) [38], closely related to tumor progression [39]. HIF-1␣ is also a promoter of MDR-1 gene expression [40]. These observations suggest that one HIF-1␣-dependent pathway could be active and inducing the upregulation of MDR-1 gene expression in tuberous sclerosis complex mutant cells through nonrepressed mTOR function.
104
PEDIATRIC NEUROLOGY
Vol. 30 No. 2
Figure 3. Schematic representation of MDR-1 and MRP-1 transporters.
In tuberous sclerosis, either of the alternative mechanisms described above could occur according to the hypothesis of a “constitutive expression” of multidrug transporters in cortical tuber abnormal cells, as proposed by Sisodiya et al. [14] in brain developmental malformations with refractory epilepsy. Our current and previous results are the only available investigations related to multidrug resistance protein expression in cortical tuber cells from epileptogenic brain lesions of patients with tuberous sclerosis; however, whether the TSC1 or TSC2 genes play any regulatory role in MDR-1 or MRP-1 gene expression still remains to be elucidated. Hypothetically, we could assume that the abnormal nature of balloon cells and dysplastic neurons evident in cortical tubers are important features that could support the presence of MDR-1 or MRP-1 in such cells as a constitutive expression of these genes. However, the presence of MDR-1 and MRP-1 proteins observed in some normal cells from the same cortical tuber lesions of our patients is in agreement with a potential mechanism of inducible expression of the multidrug resistance proteins, likely secondary to persistent and repetitive uncontrolled seizures, as demonstrated in experimental epileptic models. We believe that both mechanisms may play a critical role in the earlier development of the refractory epilepsy phenotype in patients with tuberous sclerosis. Functionally, the MDR-1 protein has been shown to elicit extrusion of a variety of hydrophobic, often cationic or neutral substrates, immunosuppressant drugs including rapamycin [41], and also planar lipophilic molecular structures, which are the most common structures of the antiepileptic drugs currently used [42]. MRP-1 protein acts as a transporter of several anionic residues of exogenous or endogenous compounds, and conjugates with glutathione, or epoxides and glucuronides, which are the major metabolic pathway of carbamazepine and lamotrigine [42]. The pattern observed of highly positive immunostaining for MDR-1 and MRP-1 proteins, in different cells of the epileptogenic cortical tubers, may result in a wide spec-
trum of refractory cells as a “pharmacoresistant cellular network” playing a critical role in the development of refractory epilepsy in tuberous sclerosis. The sum of functional properties of both proteins can confer a higher multidrug resistance phenotype to tuberous sclerosis (Fig 3). These results do not explain why the cortical tuber lesions are epileptogenic [12], but perhaps they may help to elucidate why these epileptogenic cortical tubers are refractory to antiepileptic drugs. Our results suggest that brain expression of both MDR-1 and MRP-1 transporters in the epileptogenic cortical tuber can be related to the refractory epilepsy phenotype observed in tuberous sclerosis. References [1] van Slegtenhorst M, Nellist M, Nagelkerken B, et al. Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products. Hum Mol Genet 1998;7:1053-7. [2] The European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of tuberous sclerosis gene on chromosome 16. Cell 1993;75:1305-15. [3] Jin F, Wienecke R, Xiao GH, Maize JC, Jr, DeClue JE, Yeung RS. Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region. Proc Natl Acad Sci USA 1996;93:9154-9. [4] Orimoto K, Tsuchiya H, Kobayashi T, Matsuda T, Hino O. Suppression of the neoplastic phenotype by replacement of the Tsc2 gene in Eker rat renal carcinoma cells. Biochem Biophys Res Commun 1996;219:70-5. [5] Bush JA, Li G. Cancer chemoresistance: The relationship between p53 and multidrug transporters. Int J Cancer 2002;98:323-30. [6] Juliano RL, Ling VA. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochem Biophys Acta 1976;445:152-62. [7] Gottesman MM, Pastan I. The multidrug transporter, a doubleedge sword. J Biol Chem 1988;263:12163-6. [8] Cole SP, Deeley RG. Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP. Bioessays 1998;20: 931-40 [Review]. [9] McClintock WM. Neurologic manifestations of tuberous sclerosis complex. Curr Neurol Neurosci Rep 2002;158-63 [Review]. [10] Curatolo P, Verdecchia M, Bombardieri R. Tuberous sclerosis complex: A review of neurological aspects. Eur J Paediatr Neurol 2002;6:15-23.
Lazarowski et al: MDR-1 and MRP-1 in Tuberous Sclerosis 105
[11] Avellino AM, Berger MS, Rostomily RC, Shaw CM, Ojemann GA. Surgical management and seizure outcome in patients with tuberous sclerosis. J Neurosurg 1997;87:391-6. [12] Crino PB, Miyata H, Vinters HV. Neurodevelopmental disorders as a cause of seizures: Neuropathologic, genetic, and mechanistic considerations. Brain Pathol 2002;12:212-33. [13] Lazarowski A, Sevlever G, Taratuto A, Massaro M, Rabinowicz A. Tuberous sclerosis associated with MDR1 gene expression and drug-resistant epilepsy. Ped Neurol 1999;21:731-4. [14] Sisodiya SM, Lin WR, Harding BN, Squier MV, Thom M. Drug resistance in epilepsy: Expression of drug resistance proteins in common cause of refractory epilepsy. Brain 2002;125:22-31. [15] Tishler DM, Weinberg KI, Hinton DR, Barbaro N, Annett GM, Raffel C. MDR1 gene expression in brain of patients with medically intractable epilepsy. Epilepsia 1995;36:1-6. [16] Zhang L, Ong W, Lee T. Induction of P-glycoprotein expression in astrocytes following intracerebroventricular kainate injection. Exp Brain Res 1999;126:509-16. [17] Lazarowski A, Girardi E, Ramos A, Garcı´a-Rivello H, Brusco A. MDR-1 gene expression (Pgp-170) in different brain areas in an experimental epilepsy model [Abstract]. J Epilepsy Clin Neurophysiol 2002;8:101. [18] Seegers U, Potschka H, Lo¨scher W. Expression of the multidrug transporter P-glycoprotein in brain capillary endothelial cells and brain parenchyma of amygdala-kindled rats. Epilepsia 2002;43:675-84. [19] Rizzi M, Caccia S, Guisso G, et al. Limbic seizures induce P-glycoprotein in rodent brain: Functional implications for pharmacoresistance. J Neurosci 2002;22:5833-9. [20] Kwan P, Sills GJ, Butler E, Gant TW, Meldrum BS, Brodie MJ. Regional expression of multidrug resistance genes in genetically epilepsy-prone rat brain after a single audiogenic seizure. Epilepsia 2002;43: 1318-23. [21] Lazarowski A, Girardi E, Ramos AJ, Garcı´a-Rivello H, Brusco A. Neuronal MDR-1 gene encoded P-glycoprotein (P-170) expression in 3-mercaptopropionic acid-induced seizures in rats. Epilepsia 2002; 43(Suppl. 7):11-12. [22] Sullivan GF, Yang JM, Vassil A, Yang J, Bash-Babula J, Hait WN. Regulation of expression of the multidrug resistance protein MRP1 by p53 in human prostate cancer cells. J Clin Invest 2000;105:1261-7. [23] Fukushima Y, Oshika Y, Tokunaga T, et al. Multidrug resistance-associated protein (MRP) expression is correlated with expression of aberrant p53 protein in colorectal cancer. Eur J Cancer 1999;35:935-8. [24] Oka M, Kounoura K, Narasaki F, et al. P-glycoprotein is positively correlated with p53 protein accumulation in human colorectal cancers. Jpn J Cancer Res 1997;88:738-42. [25] Thottassery JV, Zambetti GP, Arimori K, Schuetz EG, Schuetz JD. p53-dependent regulation of MDR1 gene expression causes selective resistance to chemotherapeutic agents. Proc Natl Acad Sci USA 1997; 94:11037-42. [26] Linn SC, Honkoop AH, Hoekman K, van der Valk P, Pinedo HM, Giaccone G. p53 and P-glycoprotein are often co-expressed and are
106
PEDIATRIC NEUROLOGY
Vol. 30 No. 2
associated with poor prognosis in breast cancer. Br J Cancer 1996;74: 63-8. [27] Ralhan R, Swain RK, Agarwal S, et al. P-glycoprotein is positively correlated with p53 in human oral pre-malignant and malignant lesions and is associated with poor prognosis. Int J Cancer 1999;84:80-5. [28] Sampath J, Sun D, Kidd VJ, et al. Mutant p53 cooperates with ETS and selectively up-regulates human MDR1 not MRP1. J Biol Chem 2001;276:39359-67. [29] Zhan M, Yu D, Lang A, Li L, Pollock RE. Wild type p53 sensitizes soft tissue sarcoma cells to doxorubicin by down-regulating multidrug resistance-1 expression. Cancer 2001;92:1556-66. [30] Kawaguchi K, Oda Y, Nakanishi K, et al. Malignant transformation of renal angiomyolipoma: A case report. Am J Surg Pathol 2002;26:523-9. [31] Kim SK, Wang KC, Cho BK, et al. Biological behavior and tumorigenesis of subependymal giant cell astrocytomas. J Neurooncol 2001;52:217-25. [32] Lantuejoul S, Ferretti G, Negoescu A, Parent B, Brambilla E. Multifocal alveolar hyperplasia associated with lymphangioleiomyomatosis in tuberous sclerosis. Histopathology 1997;30:570-5. [33] Cocker HA, Pinkerton CR, Kelland LR. Characterization and modulation of drug resistance of human paediatric rhabdomyosarcoma cell line. Br J Cancer 2000;83:338-45. [34] Kenerson HL, Aicher LD, True LD, Yeung RS. Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. Cancer Res 2002;62:5645-50. [35] Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC, Blenis J. Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)mediated downstream signaling. Proc Natl Acad Sci USA 2002;99: 13571-6. [36] Onda H, Crino PB, Zhang H, et al. Tsc2 Null murine neuroepithelial cells are a model for human tuber giant cells, and show activation of an mTOR pathway. Mol Cell Neurosci 2002;21:561-74. [37] Grunwald V, DeGraffenried L, Russel D, Friedrichs WE, Ray RB, Hidalgo M. Inhibitors of mTOR reverse doxorubicin resistance conferred by PTEN status in prostate cancer cells. Cancer Res 2002;62: 6141-5. [38] Hudson C, Liu M, Chiang G, et al. Regulation of hypoxiainducible factor 1␣ expression and function by the mammalian target of rapamycin. Mol Cell Biol 2002;22:7004-14. [39] Semenza G. HIF-1 and tumor progression: Pathophysiology and therapeutics. Trends Mol Med 2002;8(Suppl. 4):S62-7 [Review]. [40] Comerford K, Wallace T, Karhausen J, et al. Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res 2002;62:3387-94. [41] Sacyshyn BR, Bowen-Yacyshyn MB, Pilasky LM. Inhibition by rapamycyn of P-glycoprotein 170-mediated export from normal lymphocytes. Scand J Immunol 1996;43:449-55. [42] Levy RH, Mattson RH, Meldrum BS. Antiepileptic drugs, 4th ed. New York: Raven Press, 1995.