Molecular forms of acetyl- and butyrylcholinesterase in human glioma

ELSEVIER

Neuroscience Letters 206 (1996) 173-176

NEUROSCI LETTERS

Molecular forms of acetyl- and butyrylcholinesterase in human glioma J a v i e r S ~ i e z - V a l e r o a, G a b r i e l a P o z a - C i s n e r o s b, C e c i l i o J. V i d a l a,* aDepartamento de Bioqu£micay Biologla Molecular A, Edificio de Veterinarla, Universidad de Murcla, Apdo 4021, 30071 Murcla, Spain bServicio de Medicina Interna, Hospital Virgen de la Arrixaca, Murcia, Spain Received 5 January 1996; revised version received 10 February 1996; accepted 10 February 1996

Abstract

Specimens of astrocytorna, oligodendroglioma and medulloblastoma were sequentially extracted with saline and saline-Triton X-100 buffers. Acetyl- (ACHE) and butyrylcholinesterase (BuChE) activities were assayed in the soluble fractions, these being further analyzed to establish the distribution of molecular forms. All the tumors tested showed AChE and BuChE activities, the measured AChE/BuChE ratios being unrelated to the malignant grading. Hydrophilic and amphiphilic AChE and BuChE tetramers, amphiphilic AChE dimers and monomers, and hydrophilic BuChE monomers were identified in all the tumors analyzed. The amphiphilie behavior of the enzyme forms was assessed by sedimentation analysis and hydrophobic chromatography on phenyl-Agarose. A small fraction of glioma AChE monomers was released as, or transformed into, hydrophilic forms by incubation with phosphatidylinositol-specific phospholipase C (PIPLC). These data suggest that AChE monomers bearing distinct hydrophobic domains coexist in human glioma.

Keywords: Acetylcholinesterase; Butyrylcholinesterase; Human glioma and medulloblastoma; Hydrophilic and amphiphilic forms

Gliomas are brain tumors derived from various types of glial cells of neuroectodermal origin. They are subdivided according to their cellular differentiation into astrocytic, oligodendrocytic, ependimal or mixed composition. Intracranial tumors such as meningioma, glioma, neuroblastoma, neurinoma, angioblastoma, hemangioma and craniofaringioma express cholinesterase (ChE) activity [16,17]. Other cancerous cells as hepatoma [9] and ovarian carcinoma [ 14,18] also show ChE activity. Cholinesterases hydrolyze acetylcholine and other choline-esters. Vertebrates possess two ChEs: acetylcholinesterase (ACHE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8). AChE and BuChE differ in substrate specificity and reactivity with inhibitors [8]. Both ChEs exist as polymers and monomers of glycosylated catalytic subunits. Globular AChE and BuChE forms are abundant in the CNS where they are found as amphiphilic (G A) and hydrophilic (G H) species [2,8]. The amphiphilic AChE tetramers (G4A) from mammalian brain consist of two pairs of catalytic subunits linked to a hydrophobic 20-kDa polypeptide anchor [8]. Some amphiphilic AChE dimers * Corresponding author. Tel.: +34 68 307100, ext. 2911; fax: +34 68 364147/363963; e-mail: [email protected].

(G2 A) contain a C-terminal-linked glycosylphosphatidyl

inositol (GPI) which is removed by phosphatidylinositolspecific phospholipase C (PIPLC) [1,3,13]. Other AChE dimers maintain amphiphilicity after incubation with PIPLC and/or alkaline hydroxylamine. The exact nature of their hydrophobic domain is unknown. We report here results on the solubilization of ChEs in human astrocytoma, oligodendroglioma and medulloblastoma and on the amphiphilic or hydrophilic properties of the AChE and BuChE forms. The possible occurrence of a GPI linkage in the enzyme forms has also been investigated. Intracranial tumors were obtained at surgery, diagnosed by standard pathological techniques and stored in liquid nitrogen. Cholinesterase levels were investigated in six astrocytomas, with variable malignant grade, in two anaplastic oligodendrogliomas and in a medulloblastoma, a tumor derived from undifferentiated neuroectodermal cells. Samples were sequentially extracted 0 0 % w/v) with Trissaline (SB) and Tris-saline-Triton X-100 (STB) buffers to release the loosely- (S1) and tightly-bound ($2) ChEs, respectively (Table 1). AChE and BuChE activities were assayed in the soluble fractions. Cholinesterase forms were separated by centrifugation on 5-20% linear sucrose gradients made with 0.5% Triton X-100 or with 0.5% Brij 96.

0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All fights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 2452-4

174

J. Stlez- Valero et al. / Neuroscience Letters 206 (1996) 173-176

The amphiphilic behavior of the forms was assessed by their distinct migration in gradients made with Triton X100 or with Brij 96 [12]. Moreover, amphiphilic and hydrophilic ChEs were separated by hydrophobic chromatography, the unbound and bound forms being later identified by sedimentation analysis [ 12]. Human glioma and medulloblastoma displayed AChE and BuChE activities. The AChE/BuChE ratios were variable and unrelated with their malignant grading (Table 1), and this agrees with previous reports [11,17]. About 1520% of the tumor AChE and BuChE were released with SB (S1), and a further 4 0 - 6 0 % with STB ($2). The saline buffer's ability to extract BuChE in glioma was lower than in meningioma [13], but higher than in brain [12]. This suggests that BuChE may arise from glial and non-glial cells, although the glial origin of brain BuChE is widely accepted [6]. Sedimentation analysis of the supernatant $I in gradients with Brij 96 showed abundant (34H (11.0 S) and minor G4A (9.5 S), G2A (4.0-4.5 S) and G1A (2.5-3.0 S) AChE forms. Principal G4A and residual G2A and GI A species were identified in $2 (Table 1). As regards BuChE, abundant G4H (11.5-12.0S) and G1H (4.5-4.0S) forms were found in SI, and G4A (9.5-10.0 S) in $2. A typical profile of the ChE forms in mixtures of S 1 + S 2 from an astrocytoma (case 2) is outlined in Fig. 1, and in the separated S1 and $2 fractions in Fig. 2. Phenyl-Agarose chromatography revealed that one-half of the ACHE, and most (90%) of the BuChE activity in $1 from an astrocytoma freely passed through the hydrophobic matrix. In mixtures of $1 + $2, 75% of AChE and 25% of BuChE were bound to phenyl-Agarose. G4H and G] H ACHE, and abundant G4H

and G1H BuChE forms were identified in the non-retained fraction. About 60% of the bound AChE and BuChE eluted with Triton X-100. G4A, G2A and Gt A ACHE, and (34A BuChE forms were found in the eluted fraction (Fig. 1). The greater extractability of BuChE with saline buffer in meningioma [13] than in glioma or brain [12] probably reflects the higher G4H/G4A ratio in the former. The lack of G4H and G4A AChE and the small content of G4A BuChE in meningioma [13], compared with glioma, may be useful in establishing the origin of these tumors. The profiles of AChE forms in glioma and brain differ in a greater contribution of Gt A AChE in the former, probably reflecting a specific biosynthetic pathway in the tumor cells. Although glioma is richer in G4H BuChE than is brain, the absence of major differences between the profiles of BuChE forms in S] and $2 from the two sources supports the glial origin of the brain enzyme. The possible occurrence of a GPI anchor in the AChE forms was investigated by exposing an astrocytoma suspension in SB to PIPLC. A slight stimulation (15-20%) of the AChE activity was measured in the homogenate after incubation with PIPLC, this effect being attributed to some structural modification induced in the enzyme by the loss of the GPI anchor. A comparison of the ChE forms extracted from an astrocytoma with SB, without and with PIPLC, revealed a higher content of the 4.2 S A C h E in samples exposed to the phospholipase (profiles not shown). This suggested that a small fraction of the G1A (2.5 S) A C h E was released as, or transformed into, hydrophilic species. The conversion of some Gi A AChE into Gi H forms was further assessed by incubating the extract $1 with the phospholi-

Table 1 ChEs in human astrocytoma, oligodendroglioma and medulloblastoma

Case Tumor (WHO classification)

1 2 3 4 5 6 7 8 9

Ast. (LG) Ast. (LG) Ast. (LG) Ast. (LG) Ast. (LG-HG) Ast. (HG) Oli. (HG) Oli. (HG) Med. (HG)

AChE activity (U/ml)

BuChE activity (U/ml)

Protein (mg/ml)

S1

S2

SI

$2

S1

$2

0.53 0.36 1.53 0.15 0.34 0.68 1.20 2.69 0.93

3.15 2.31 8.69 1.09 0.71 2.25 2.80 14.89 2.95

0.82 2.71 2.31 5.28 1.23 2.30 0.90 5.80 0.46

0.69 3.03 2.64 2.57 1.04 3.67 0.77 14.01 0.33

5.0 2.9 6.7 6.2 2.1 1.7 4.9 4.8 4.5

3.9 2.2 3.8 4.6 2.2 1.3 2.0 2.0 2.8

ACHE/ BuChE ratio

2.44 0.28 2.06 0.16 0.46 0.80 2.49 0.89 4.72

AChE forms (%)

BuChE forms (%)

G4H

G4A

G2A

GI A

G4H

G4A

GI H

5 8 4 7 9 5 19 5 7

84 55 82 33 64 55 43 82 58

5 35 7 17 13 5

11

22 14 25 20 23 25 13 30

65

17

36 42 70 20 27 55 15 48

41 45 17 55 51 25 65 34

18 23 13 13 25 22 20 20 18

The tumors (Ast., astrocytoma; Oli., oligodendroglioma; Med., medulloblastuma) are classified into low (grade I or I1; LG) and high malignant grading (grade IIl or IV; HG), as recommended by the World Health Organization (WHO). Frozen specimens were thawed at 4*(2 and sequentially extracted with 1 M NaCl, 50 mM MgCI2, 10 mM Tris (pH 7.0) (Tris-saline buffer, SB) containing a mixture of antiproteinases [12] to release the soluble and loosely-bound ChEs (S1), and later with 1% w/v Triton X-100 and antiproteinases in saline buffer (Tris-saline-Triton X-100 buffer, STB) to detach the tightly-bound ChEs ($2). Soluble fractions S 1 and S2 were recovered by centrifugation at 100 000 x g, 1 h at 4°C. AChE and BuChE activities and protein content were determined as reported previously [12]. ChE activity is expressed in/~mol of substrate hydrolyzed/h per ml (U/ml). AChE/BuChE ratios were calculated by adding the enzyme activities in S 1 and S2. Molecular forms of AChE and BuChE were separated by sedimentation analysis as described in Fig. 1 and assigned to individual forms according to previous reports [12,13]. The relative content of the separated enzyme forms was calculated from the percentages of each form in the sedimentation profiles of St and S2 and from the amount of enzyme solubilized in each fraction. All tumors contained variable amounts of G2 ACHE, although its relative proportion could not always be calculated.

J. Stiez-Valero et al. I Neuroscience Letters 206 (1996) 173-176

AChE -

BuChE 'Sl+S2

C

P

4,

4,

C

P Sl+S2

4,

4,

- 16

,l | ~

I

I A H

i

i

,

0

UF _o

UF

¢:3

4,

4,

4,

4,

175

the enzyme forms. Nevertheless, our results suggest that glioma and medulloblastoma contain two types of G1A forms with different sensitivity to PIPLC. Whether the distinct amphiphilic domains in AChE monomers serve for binding them to different cells or to~distinct membranes in a cell merits further investigation. As regards the origin of the AChE forms in glioma, it is worth mentioning that G4A, G4rl, G2A and GI A species are synthesized in COS cells transfected with a cDNA which, in rat brain, encodes the subunits for AChE tetramers [8]. The identification of these forms in glioma implies that neuronal and glial cells produce a full set of AChE species. Moreover, three mRNAs which encode AChE subunits have been identified in various tumor cell lines [5]. AChE

'P

>

C

~5

4, 4, ~,,

85

uJ

r-

I

,

I

,

I

I

,



BF $

4,

4,

,

I

o

4,

I

I

20

30

--

3

I

I

I

I

I

20

30

40

C

P

4,

4,

$1 30

0

,1,

4,

pase. Again, the percentage of the 4.2 S AChE increased by PIPLC (Fig. 2). Similar results were obtained by exposing St from an oligodendroglioma or a medulloblastoma to PIPLC. The strong inactivation of the GI g AChE by alkaline hydroxylamine prevented us for further investigating its efficiency for cleaving the GPI anchor in

$

S'I

$

S'l.

A H

Fraction Fig. 1. Separation of amphiphilic and hydrophilic ChE forms in human glioma. A single frozen tumor was thawed at 4°C and homogenized in 1 M NaCI, 50 mM MgCI 2, 10 mM Tris (pH 7.0) (saline buffer, SB) containing a mixture of antiproteinases [12]. A soluble fraction (S 1) deh in soluble and loosely-bound ChEs was saved by centrifugation at 100 000 x g, 1 h at 4°C. The remaining pellet was re-extracted with SB, I% Triton X-100 and antiproteinases (STB) and the Triton-soluble fraction ($2) was recovered by eentrifugation as above. Equal volumes of the supematants S 1 and S 2 were mixed (2 ml) and applied onto a phenyl-Agarose column (6-7 ml) pre-equilibrated with SB. The nonretained hydrophilic ChE eluted with SB and the amphiphilic enzyme with 2% Triton X-100 in 10 mM Tris buffer (pH 7.0). ChE forms in S 1 + S 2 and in the unbound (UF) and bound fractions (BF) were separated by centrifugation on 5-2(}% linear sucrose gradients made in SB with 0.5% Brij 96. The gradient: tubes were centrifuged at 150 000 x g, 18 h at 40C, fractions collected from the bottom and assayed for AChE and BuChE [12]. The individual enzyme forms were identified in accordance with previous data [ 12,13]. Internal markers were catalase (C, 11.4 S) and alkaline phosphatase (P, 6.1 S).

15

I ¢) ¢-

4010

Sl

q)

A

I

-

P

BF -6

' lb

10

BuChE

-4

124 12

.> 85

uJ

tO

0

I

18

,

I

,

I

12

0

I I

1

,

1

,

1

S2

2

,!

4,

,

4,

50

25 •

,

H

34

z

I

I

I

10

20

30

I

4010

I

20

,

I

30

J

I

40

Fraction Fig. 2. Conversion of amphiphilic AChE monomers into hydrophilic derivatives by treatment with PIPLC. Molecular forms of AChE and BuChE in the saline extract before (S1) and after incubation with 0.8 U/ml PIPLC from Bacillus thuringiensis (Funakoshi Co. Ltd., Japan) 2 h at 36°C (S') and in the saline-Triton X-100 extract ($2). Note the increase of the shoulder containing the G2A and GI H AChE forms in samples incubated with PIPLC and the maintenance of the BuChE profiles. Sucrose gradients made with 0.5% Brij 96. Sedimentation markers as in Fig. 1.

176

J. Stiez-Valero et al. / Neuroscience Letters 206 (1996) 173-176

Whether the different forms in glioma arise from a single or from various mRNAs remains to be clarified. As regards BuChE, various forms are produced in Xenopus oocytes injected with embryonic brain, glioma or meningioma mRNAs [15]. Whether BuChE subunits in G4A, G4H, and GI H forms are encoded by several genes or by alternative splicing of a single mRNA is unknown. A unique cDNA has been found in brain, and a further sequence in glioblastoma. The tumor-specific cDNA showed various alterations and the Asp7°-Gly mutation [4], which was also detected in the atypical plasma BuChE [7]. Cholinesterases genes are amplified in cancerous and non-cancerous cells [19]. BuChE is probably involved in megakaryocyte growth, proliferation and differentiation. The fact that megakaryocytopoiesis was controlled with antisense BuChE oligonucleotides [10] offers new alternatives for slowing tumor cell proliferation. J. S~iez-Valero was supported by a scholarship from the Ministerio de Educaci6n y Ciencia of Spain. We are grateful to Drs. M. Poza and C. Piqueras, both from the Neurosurgery Unit at the Hospital Virgen de la Arrixaca in Murcia for providing tumor specimens, and Dr. F.J. Campoy for helpful discussion. [1] Bon, S., Rosenberry, T.L., and Massouli6, J., Amphiphilic, glycophosphatidylinositol-specific phospholipase C (PI-PLC)-insensitive monomers and dimers of acetylcholinesterase, Cell. Moi. Neurobiol., 11 (1991) 157-172. [2] Chatonnet, A. and Lockridge, O., Comparison of butyrylcholinesterase and acetylcholinesterase, Biochem. J., 260 (1989) 625-634. [3] Ferguson, M.A.J., Glycosyl-phosphatidylinositol membrane anchors: the tale of a tail, Biochem. Soe. Trans., 20 (1992) 243-256. [4] Gnatt, A., Prody, C.A., Zamir, R., Lieman-Hurwitz, J., Zakut, H. and Soreq, H., Expression of aitematively terminated unusual human butyrylcholinesterase messenger RNA transcripts, mapping to chromosome 3q26-ter, in nervous system tumors, Cancer Res., 50 (1990) 1983-1987. [5] Karpel, R., Ben Aziz-Aloya, R., Sternfeld, M., Ehdich, G., Ginzberg, D., Tarroni, P., Clementi, F., Zakut, H. and Soreq, H., Expression of three alternative acetylcholinesterase messenger RNAs in human tumor cell lines of different tissues origins, Exp. Cell Res., 210 (1994) 268-277.

[6] Koelle, G.B., Cytological distribution and physiological functions of cholinesterases, Handb. Exp. Pharmacol., 15 (1963) 187-298. [7] La Du, B.N., Bartels, C.F., Nogueira, C.P., Hajra, A., Lightstone, H., Van der Spek, A. and Lockridge, O., Phenotypic and molecular biological analysis of human butylcholinesterase variants, Clin. Biochem., 23 (1990) 423-431. [8] Massoulit, J., Pezzementi, L., Bon, S., Krejci, E. and Vallette, F.M., Molecular and cellular biology of cholinesterases, Prog. Neurobiol., 41 (1993) 31-91. [9] Osada, J., Ltpez-Miranda, J., Sastre, J. and Ordovfis, J.M., The human hepatoma cell line, HepG2, secretes functional cholinesterase, Biochem. Mol. Biol. Int., 33 (1994) 1099-1105. [10] Patinkin, D., Seidman, S., Eckstein, F., Benseler, F., 7_,akut, H. and Soreq, H., Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro, Mol. Cell Biol., 10 (1990) 6046---6050. [I1] Razon, N., Soreq, H., Roth, E., Bartal, A. and Silman, I., Characterization of activities and forms of cholinesterases in human primary brain tumors, Exp. Neurol., 84 (1984) 681-695. [12] Sfiez-Valero, J., Tomel, P.L., Mufioz-Delgado, E. and Vidal, C.J., Amphiphilic and hydrophilic forms of acetyl- and butyrylcholinesterase in human brain, J. Neurosci. Res., 35 (1993) 678-689. [13] S~iez-Vaiero, J. and Vidal, C.J., Monomers and dimers of acetylcholinesterase in human meningioma are anchored to the membrane by glycosylphosphatidylinositol, Neurosei. Lett., 195 (1995) 101-104. [14] Soreq, H. and Zakut, H., Amplification of butyrylcholinesterase and acetylcholinesterase genes in normal and tumor tissues: putative relationship to organophosphorous poisoning, Pharmacol. Res., 7 (1990) 1-7. [15] Soreq, H., Zevin-Sonkin, D. and Razon, N., Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species, EMBO J., 3 (1984) 1371-1375. [16] Stieger, S., Biatikofer, P., Wiesmann, U.N. and Brodbeck, U., Acetylcholinesterase in mouse neuroblastoma NB2A cells: analysis of production, secretion and molecular forms, J. Neurochem., 52 (1989) 1188-1196. [17] Wolleman, M. and Zoltan, L., Cholinesterase activity of cerebral tumors and tumorous cysts, Arch. Neurol., 6 (1962) 161-167. [18] Zakut, H., Ehrlich, G., Ayaion, A., Prody, C.A., Malinger, G., Seidman, S., Ginzberg, D., Kehlenbach, R. and Soreq, H., Acetylcholinesterase and butyrylcholinesterase genes coamplify in primary ovarian carcinomas, J. Clin. Invest., 86 (1990) 900-908. [19] Zakut, H., Lapidot-Lifson, Y., Beeri, R., Bailin, A. and Soreq, H., In vivo gene amplification in non-cancerous cells: cholinesterase genes and oncogenes amplify in thrombocytopenia associated with lupus erythematosus, Mutat. Res., 276 (1992) 275-284.