Neuroscience Letters, 108 (1990) 11 16 11 Elsevier Scientific Publishers Ireland Ltd.

NSL 06535

Type-specific expression of protein kinase C isozymes in CNS tumor cells

Sadashi Shimosawa 1, Takahisa Hachiya l, Masatoshi Hagiwara I , Nobute ru Usuda 3, Kenichiro Sugita 2 and Hiroyoshi Hidaka 1

1Department of Pharmacology and 2 Neurosurgery, Nagoya University School of Medicine, Nagoya (Japan) and 3Department of Anatomy, Shinshu University School of Medicine, Matsumoto (Japan)

(Received 16 August 1989; Accepted 21 August 1989)

Key words: Protein kinase C isozyme; Neuroblastoma cell; Glioblastoma cell; Monoclonal antibody

We examined specific expression of protein kinase C (PK-C) isozymes in cultured human glial and neur- onal cell lines, using type-specific monoclonal antibodies MC-Ia, -2a, and -3a (Hidaka H. et al., J. Biol. Chem., 263 (1988) 4523~,526). Immunoblotting experiments revealed that a 80 kDa band of three kinds of glioblastoma cells (A-172, SK-MG-1, SK-MG-4) was stained with MC-3a, whereas that of neuroblas- toma cells (SK-N-MC) reacted with MC-2a. Immunoenzymetric assay showed that glioblastoma cells (A-172, SK-MG-I, SK-MG-4) contained 127.6_+ 14.4, 248.8_+ 38.5 and 148.5 _+ 35.8 ng/mg protein of type III, respectively, while neuroblastoma cells (SK-N-MC) contained 389.5_+ 20.7 ng/mg protein of type II. These results suggest that PK-C isozymes may be specifically expressed, depending on types of central nervous system (CNS) tumor cells.

Protein kinase C (PK-C) was first reported by Nishizuka and associates to be a single enzyme [15]. Huang et al. identified 3 PK-C types, using hydroxylapatite chro- matography [7], and Hidaka et al. obtained evidence for the immunological differ- ences in the 3 fractions [5]. cDNA cloning and sequence analysis of PK-C suggested the existence of several forms of this kinase, and structure and genetic identity of each type were clarified [2, 6, 9, 11-14]. Each PK-C isozyme was found to be present in different brain regions, cell types, subcellular locations, and developmental stages of the brain [1, 5, 10, 18, 19]. Although PK-C is highly abundant in the central nervous system (CNS) [3, 10, 18], the regional distribution has not been fully characterized. A detailed description of the distribution of PK-C in the brain would provide impor- tant information that would aid in elucidating the diverse effects of PK-C. The CNS mainly consists of neuronal and glial cells. We examined PK-C isozymes in the glio- blastoma cells and neuroblastoma cells, using monoclonal antibodies (MC-la, -2a and -3a) prepared by injecting the purified PK-C into mice, as described by Hidaka

Correspondence." H. Hidaka, Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Nagoya 466, Japan.

0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

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et al. [5]. Human glioblastoma cells (A-172, SK-MG-1, SK-MG-4) [16] and human neuroblastoma cells (SK-N-MC) [16] were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (Gibco Labs, Grand Is- land, NY) penicillin G (100 U/ml), and streptomycin (50/tM) at 37"C in a humidified atmosphere of 5% CO2, 95% air. The confluent layer of these cells was scraped out into 4 ml of buffer B (25 mM Tris-HC1 pH 7.3, 1 mM EGTA, 50 mM 2-mercaptoeth- anol (2-ME). 0.001% leupeptine, and 10% (v/v) glycerol). After sonication, whole homogenates were centrifuged at 100,000 g for 30 min at 4°C to obtain the cytosolic fraction. Twenty / tg of protein in the cytosolic fraction were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) in 10% gels, fol- lowed by transblotting the protein on a nitrocellulose membrane (Bio-Rad Labs, Richmond, CA). After transfer, immunoreactive protein was stained using an ABC Kit (Vector Labs, Burlingame, CA), and visualized with 0.06% (w/v) 4-chloro-1- naphtol and 0.02% H202, as described by Watanabe et al. [17].

The levels of PK-C isozymes in the cultured cells were determined by immunoenzy- metric assay. Monoclonal antibodies were placed in wells of microtiter plates. After an overnight incubation, the wells were washed in PBS, incubated with blocking buf- fer and washed again. The confluent layer was scraped out into 2 ml of homogenate buffer (phosphate-buffered saline (PBS) containing 0.1% NAN3, 0.00t% leupeptine, 0.05% Tween-20, 5 mM EDTA and 1 mM MgC12). After sonication, whole homoge- nates were centrifuged at 100,000 g for 30 min at 4~C, and the process repeated. The supernatants were passed through M I N I S A R T N M L (pore size 0.45 pm Sartorius GmbH, G6ttingen, F.R.G.). The preparations were incubated and washed with PBS. Polyclonal antibody against the C-terminal peptide of PK-C, (named PC-40, Hagi- wara et al. submitted), conjugated with horseradish peroxidase (HRP), was added and HRP activity bound to the well was assayed. PK-C activity in the DEAE fraction

80 KDa. '~"

s 1 2 3 4

M C - l a MC-2a MC-3a

80 KDa. "~ 80 KDa'"~

S 1 2 3 4 $ 1 2 3 4

Fig. I. lmmunoblot analysis of PK-C isozymes in glioblastoma and neuroblastoma cells. Fractions from cultured glioblastoma and neuroblastoma cells were subjected to SDS PAGE and transferred to a nitro- cellulose membrane. Immunoblot analysis was done with monoclonal antibody against type I (MC-la), type I1 (MC-2a), or type III (MC-3a) of PK-C. The molecular mass in kDa of the standards are indicated by arrows: Tissue sample from rabbit brain including PK-C type 1, II, and III (lane S), A-172 cells (20 /Lg protein) (lane 1), SK-MG-I cells (20 pg protein) (lane 2), SK-MG-4 cells (20 #g protein) (lane 3), and SK-N-MC cells (20 Itg protein) (lane 4).

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was assayed by measuring the incorporation of 32p from [7-32p]ATP into Histone III-

S by a modified procedure of Hidaka and Tanaka [4]. Cultured cells on slide glass (Lab-Tec Chamber Slide, Nunc, Inc., Naperville, IL) were stained by the indirect

immunofluorescent method described by Ito et al. [8]. Immunoblot t ing analysis in glioblastoma and neuroblastoma cells using type spe-

cific monoclonal antibodies revealed that only MC-2a recognized the 80 kDa protein in SK-N-MC cells, and only MC-3a recognized the 80 kDa protein in A-172 cells, SK-MG-I cells, and SK-MG-4 cells (Fig. 1). The reaction was specific to the protein and no other immunoreactive proteins were observed. As a negative control, we used normal mouse serum instead of monoclonal antibodies and no cell line stained (data not shown). Quantitative estimations of PK-C isozymes in glioblastoma and neuro- blastoma cells are shown in Table I. The M C - I a reacting protein was not detected in any cell line. The amount of MC-2a reacting protein in SK-N-MC cells was 389.5 + 20.7 (ng/mg protein) and that of MC-3a reacting protein in A-172, SK-MG- 1, and SK-MG-4 cells was 127.6+ 14.4, 248.8+38.5, and 148.5+35.8 (ng/mg pro- tein), respectively. Results of immunoblott ing experiments and immunoenzymetric

assay were in good accord. To determine whether or not the MC-2a and MC-3a reacting proteins were PK-C,

we attempted to isolate PK-C from a large quantity of cultured A-172 cells. The elu- tion pattern of PK-C activity is shown in Fig. 2. As is evident from the DEAE-cellu- lose chromatography profile, the PK-C activity (closed circles) as characterized by Ca 2+ and phosphatidylserine dependent [7-32p]ATP incorporation into Histone III-S

was eluted at a concentration of 0.1-0.16 M of NaCI. A major peak of PK-C activity appeared in Fractions 15-19, which were pooled (0.21 mg protein/ml) and PK-C ac- tivity contained in A-172 cells was estimated as 414 (pmol/min/mg protein). The PK- C rich fraction was subjected to hydroxylapatite chromatography, but the elution pattern could not be separated into three distinct fractions because of a small amount of sample (data not shown). Though PK-C isozymes can be separated by hydroxyla-

TABLE I

QUANTITATIVE ESTIMATION OF PROTEIN KINASE C ISOZYMES IN GLIOBLASTOMA AND NEUROBLASTOMA CELLS

Quantitative estimation of PK-C isozymes was carried out by the sandwich method using monoclonal antibodies specifically binding to N-terminals of PK-C, as the capture antibodies, and polyclonal antibody binding to C-terminal of PK-C as the sandwich cover. The values (mean_+S.D.) were obtained from 4 experiments, n.d., non-detectable.

MC- 1 a MC-2a MC-3a (ng/mg protein) (ng/mg protein) (ng/mg protein)

A-172 n.d. n.d. 127.6+ 14.4 SK-MG- 1 n.d. n.d. 248.8 _+ 38.5 SK-MG-4 n.d. n.d. 148.5_+35.8 SK-N-MC n.d. 389.5_+20.7 n.d.

14

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Fig. 2. PK-C activity in A- 172 cells. The cytosolic fraction of A- 172 cells was applied to a DEAE-cellulose column (1 x 3 cm) equilibrated in buffer B, the column was washed with 100 ml of buffer B, the bound enzyme was eluted with a linear gradient (50 ml) from 0 to 0.4 M NaCI in the same buffer, and 1 ml frac- tions were collected. PK-C activity was assayed by measuring the incorporation of 32p from [7-32P]ATP into Histone I II-S: protein kinase activity with C a 2+ and phosphatidylserine ( H ) , protein kinase activity without Ca 2 + and phosphatidylserine (C) C:, ), NaCI concentration (...).

Fig. 3. PK-C isozymes in A-172 cells stained by indirect immunofluorescence techniques. The cells were incubated with each monoctonal antibody against PK-C type I (MC-la) (1), type II (MC-2a) (2), and type Il l (MC-3a) (3), and normal mouse ascites (4), then stained. Scale for atl panels = 4 0 ,um.

15

pa t i te co lumn c h r o m a t o g r a p h y , this m e t h o d is no t sui table for rou t ine analysis with

l imited amoun t s o f sample. P K - C in A-172 cells was also invest igated using an indir-

ect immunof luorescen t technique with M C - l a , -2a, and -3a (Fig. 3). M C - 3 a demon-

s t ra ted a cytosol ic d i s t r ibu t ion o f the immunoreac t ive PK-C, while M C - l a and M C -

2a p r o d u c e d only a faint immunof luorescence staining. Cel lu lar immunoreac t iv i ty

with M C - 3 a was due to s ta ining o f the cy top lasm ra ther than the per iphery o f the

nucleus.

We also examined h u m a n e p e n d y m o m a cells (KMS-I I -104) [16] s ta ined with MC-

3a. Immunoenzyme t r i c assay revealed contents o f 161.3+48.5 (ng/mg prote in) o f

M C - 3 a react ing prote in . Other isozymes o f PK-C were not evident in this assay

(unpubl i shed data) . M e n i n g i o m a did not con ta in any pro te in react ing with the anti-

bodies we used (da ta not shown).

These results suggest that P K - C isozymes have a cel l- type specificity in C N S tu-

mors , and tha t de tec t ion o f P K - C isozymes will a id in under s t and ing the diverse ef-

fects o f P K - C and also to d iagnose tumors der ived f rom glial cells, neurona l cells,

o r the meninges.

We are grateful to Dr. M. Sh ibuya and Dr. J. Yosh ida for their k ind coopera t ion ,

and to M. O h a r a for cri t ical reading o f the manuscr ip t . This invest igat ion was sup-

po r t ed in pa r t by research grants f rom the Scientific Research F u n d o f the Min is t ry

o f Educa t ion , Science and Cul ture in Japan.

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