Journal ~f the Neurological Sciences, 101 ( 1991 ) 19-23 Elsevier

19

JNS 03454

Transforming growth factor (TGF)-fi like activity of intracranial meningioma and its effect on cell growth Taizo Nitta

1,2,

Kiyoshi Sato I and Ko Okumura 2

Departments qf JNeuroh~gical SurgeD' and elmmunologv, Juntendo UniversiO', School oj' Medic#le, Tok~vo (Japan) (Received 12 June, 1990) (Revised, received 7 September, 1990) (Accepted 10 September, 1990)

Key words."Transforming growth factor (TGF); Meningioma; Brain tumors; Central nervous system; Transformation

Summary Polypeptides, characterized by their ability to confer a transformed phenotype on an untransformed indicator cell have been isolated directly from surgical specimens of intracranial meningioma by using an acid/ethanol extraction procedure. Transforming activity in meningeal cells was based on the ability to induce NRK 49F rat kidney fibroblasts to form colonies in soft agar. This polypeptide was separated by gel filtration into two fragments of 15 and 40 kilodalton (kDa) molecular weight. Among other cases of brain neoplasms, one case ofgliobtastoma muttiforme had moderate TGF-fi activity, but medulloblastoma and neurinoma had no activity. Purified TGF-fl also stimulated DNA synthesis in primary cultured meningioma cells, but no effect was seen in U 251MG human glioma cells. While the physiological function of TGF-fl is still ill-defined and the molecular character of its receptor has not been analyzed, intracranial meningiomas are noted to have TGF-fi-like activity. TGF-fi also induces the DNA synthesis of cultured meningioma cells. From these results, TGF-fi would be considered one of the growth promoting factors in meningioma.

Introduction Transforming growth factor (TGF) may be defined as polypeptides that have an ability to cause normally anchorage-dependent mesenchymal cells to form colonies in semisolid agar (Roberts et al. 1981). T G F is separated into two molecules. One is T G F - ~ which has been shown to be an acid- and heat-stable molecule with a sequence homologous to epidermal growth factor (EGF) (Marqurdt et al. 1983; Todaro 1978). The other one, TGF-fl, is a 25 kDa acidstable molecule composed of two identical polypeptide chains which are widely distributed in various neoplastic and non-neoplastic tissues (Martin et al. 1987). Interestingly, depending on the tis sue source and degree of differentiation, TGF-fi can up- and down-regulate the cellular function (Assoian 1985). Therefore, rather than being regarded as a growth promotor, TGF-fl is now considered a multipotential growth regulator that has an important role

in controling replication and differentiation (Kasid et al. 1988). Intracranial meningiomas account for about 15% of all primary brain tumors and 25 % of spinal cord tumors. They are considered to be derived from arachnoidal cap cells or related cells such as meningeal fibroblasts (Cushing and Eisenhardt 1983). It is true that meningiomas are closely related to the menings, but the primitive precursor cell and its biological Function has not been clearly defined. Moreover, ambiguous classification of angioblastic or hemangiopericytic meningiomas may cause confusion (Pitkethly et al. 1976). According to some authors, TGF-fi induces a proliferative response on mesenchymal cells and facilitates fibroblasts to produce collagen, repairing wound scars and angiogenesis (Roberts et al. 1986). Histological inspection among and around the mcningioma revealed 11o immune reaction of lymphocytes or macrophages similar to malignant glioma. Therefore, some growth promoting factor is suspected to be involved in the tumorigenesis of meningioma.

Correspondence to." Taizo Nitta, M.D., Department of Neurology, Genetics and Neurological Science, Stanford University School of Medicine, Stanford, CA 94305-5235, U.S.A. Phone: (415)725-6401; Fax: (415 ) 725-7459.

Here we have tried to purify the growth promoting factor from various intracranial tumors by an acid ethanol extraction procedure. Transforming activity was then tested by the method of colony formation on soft agar and the effect

(1(/22-510X/91,$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

2O of purified TGF-/~ upon the cultured primary meningioma cells was examined.

Materials and methods

Five histologically verified cases of intracranial meningioma and one case each of glioblastoma multiforme, medulloblastoma at cerebellar vermis and acoustic neurinoma were included as the study subjects. Patient's profile of meningioma is summarized in Table 1. Case 5 is a recurrent case of angioblastic meningioma of the right parietal convexity area. Normal rat fibroblast clone, N R K 49F to assay for TGF-/~ (Roberts et al. 1980) and a human glioma cell line U 251MG were obtained from the American Type Culture Collection (ATCC) and were suspended in Dulbecco's modified Eagle's medium (DMEM, Gibco Oriental) supplemented with 5~o fetal calf serum (FCS), 5 × 10 5 M 2-mercaptoethanol, 100 U/ml penicilin, 100 #g/ml streptomycin (Sigma Co., St. Louis, MO). Crude TGF-/~ was extracted from tumor specimens by a modification of the acid/ethanol procedure (Davoren 1962). Tumor specimens were washed vigorously with phosphate-buffered saline (PB S) and the vasculatures were trimmed with scissors as much as possible. They were thawed in 40 ml/10g tissue in a solution consisting of 375 ml 95~o (v/v) ethanol and 7.5 ml of HC1 plus 33 mg phenymethylsulfonyl fluoride (PM S F) and 1.9 mg pepstatin as protease inhibitors. The volume was adjusted to 6 0 m l / 1 0 m g tissue and the tissue was minced in a Tissumizer. After an overnight extract at 4 ° C, the mixture was centrifuged and the the residue was re-extracted for 2 h with 40 ml of a solution consisting of 375 ml 9 5 ~ ethanol, 105 ml distilled water and 7.5 ml HC1. The combined supernatants were kept in the acidic range by adjusting the pH to 8.2 with concentrated ammonium hydroxide, followed by the addition of 1 ml 2 M ammonium acetate buffer, pH 5.3 per 85 ml extract. Two volumes of cold anhydrous ethanol TABLE 1 CLINICAL DATA OF THE PATIENTS SUFFERING FROM MENINGIOMA Patient No.

Age/sex

Location a

Histologyb

1 2 3 4 5

48/M 56/F 54/F 49/M 60/F

convexity parasagittal shenoid w i n g tuberculumsella convexity

meningothelial fibroblastic transitional transitional angioblastic (recurrence)

a The location of tumor was decided from operative findings and other neuroradiologiealprocedures. b Histologicalnomenclaturewas accordingto the WHO classification.

and 4 volumes of cold anhydrous ether were immediately added, after which the mixture was allowed to stand at - 2 0 °C for 30-48 h. The resulting precipitate was collected by centrifugation and redissotved in 1 M acetic acid. After extensive dialysis at 4 °C against 0.17 M acetic acid, the samples were lyophilized and stored at - 70 ° C. The acid/ethanol extract prepared was purified by size-exclusion high-performance liquid chromatography (HPLC, Pharmacia) using the T S K 3000SWXL column (TOSO) (Nitta et al. 1989). The assay for colony growth in soft agar was carried out according to established procedures (Detarco and Todaro 1978) (Fig. 1). Briefly, samples to be tested were suspended in 0.3"o agar (Noble agar, Difco) in DMEM (3 × 1(13 cells/ml). An aliquot of the mixture was pipetted onto a 0.7-ml base layered 35-mm 3 petri dish (Falcon). Plates were then incubated at 37 °C for 2 weeks in a humidified 5 3~, CO 2 atmosphere without further feeding. At 2 weeks, 0.7 ml of a sterile solution 2-(p-iodophenyl)-3-(p-nitrophenyl))-5-phenyl tetrazolium chloride (0.5 mg/ml in water) was layered over the agar and the incubation was continued for 24 h. After removal of the excess dye solution, these plates could be scored in a bright-field microscope. A unit of soft agar colony-forming activity is defined as the amount of protein that stimulated the formation of the colony ( > 10 cells) per cm 2 in 2 weeks under the described assay conditions. To explore the effects of TGF-/~ on meningioma cells, surgical specimens of meningioma were used for proliferative assays. Surgically resected tissues were mechanically minced into 1-mm 3 pieces, followed by trypinization (0.25 ~o trypsin, Gibco) as described in previous procedures (Nitta et al. 1990). After washing twice with culture medium, they were seeded on 75-cm 3 culture flasks at a concentration of 106 cells/ml. Three or 4 days later, cells which became confluent were trypsmized and washed with PBS and were replated into 96 welt tissue culture plates (1 × 10 4 cells/well) in serum-free medium (AIM-V; Gibco) with various concentrations of purified TGF-/~ (Collaborative Research). After culturing at 37 °C in a 5j°'o CO2 incubator for 48 h, they were pulsed with 10 ~Ci [3H]thymidine (New England Nuclear). Cell proliferation was measured by [3H]thymidine uptake alter 6-h pulse by harvesting cells onto glass fiber filters with a cell harvester. Radioactivity was counted in a liquid scintillation counter. As a control, an established human glioma cell line U 251MG cells was used.

Results

Among the acid/ethanol extracts tested, 4 in 5 cases of meningioma and one case of glioblastoma multiforme had

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Fig. 1 indicate that the extract of the recurrent angioblastic meningioma (case 5) had the highest specific activity in the assay, whereas the medulloblastoma and neurinoma had no

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activity. T h e s e results s h o w that crude extracts of T G F - f l like activity could be obtained from intracranial m e n i n -

gioma. The acid/ethanol extract of meningioma was further purified by gel chromatography on a HPLC system. As Fig. 2 shows, several peaks were seen. After each eluted fraction was concentrated, transforming activity was measured by soft agar colony formation as described above. The major peak of crude extract was eluted at about

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70 k D a , but T G F - f l activity mainly resided in 2 peaks. The

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first peak of TGF-fi activity is localized at a molecular

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weight o f approximately 40 k D a . This peak contains 3 % of

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the total protein recovered from the column and 50% of the soft agar activity in the column. The latter peak appears at

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approximately 15 k D a . This peak contains 10% of the total protein and 30~o of T G F - f l activity from the column. From this elution profile and soft agar activity, 2 peaks of T G F - f l activity were noticed.

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I0 Protein, #g/ml

Meningioma(O),

Meningioma, R e c u r r e n c e ( E ] )

Besides this, we have examined whether TGF-fi m a y trigger the proliferation of "meningioma cells' or not,

Ghoblastoma M u l t i f o r m e ( - - - I - -), M e d u l i o b l a s t o m a ( I ) Neurinoma ( • ',

Fig. 1. Dilution curves of a soft agar colony, forming activity in intracranial tumors. Surgically resected tissues were extracted with acid/ ethanol and their crude extracts were assayed for TGF-fl activity. Horizontal bar shows a concentration of crude extract from various tumor samples. Vertical bar represents the number of colonies per cm-'. Number shown in meningiomas is the patient number in Table 1.

according to [ 3 H ] t h y m i d i n e incorporation m e t h o d s . A s s h o w n in Fig. 3, primary cultured cells from m e n i n g i o m a

responded to purified TGF-fl in a dose-dependent manner, s h o w i n g no influence upon U 2 5 1 M G .

M e n i n g i o m a of

transforming activity as measured by the stimulation of normal a n c h o r a g e - d e p e n d e n t rat kidney fibroblasts to form colonies in soft agar. The d o s e r e s p o n s e curves s h o w n in

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Transforming growth factor (TGF)-beta like activity of intracranial meningioma and its effect on cell growth.

Polypeptides, characterized by their ability to confer a transformed phenotype on an untransformed indicator cell have been isolated directly from sur...
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